Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter

ABSTRACT

The invention relates to a xylanase originating from a Bacillus strain. This xylanase is active over a wide range of acid and basic pH.  
     The invention also relates to new strains of microorganisms producing this xylanase and to methods for preparing this xylanase.  
     The invention also relates to a DNA molecule and to an expression vector or an integration vector containing this DNA molecule.  
     The invention also relates to uses of the latter and to compositions containing it.

[0001] The invention relates to a new xylanase. The invention alsorelates to the methods for preparing this xylanase, to the uses of thelatter and to compositions comprising it.

[0002] The invention also relates to a new strain of microorganismsproducing this xylanase and to a DNA molecule comprising the nucleotidesequence which codes for this xylanase. The invention also relates tovectors containing this DNA molecule and to strains transformed by thesevectors.

[0003] The invention also relates to the promoter derived from the genewhich codes for Bacillus pumilus PRL B12 xylanase and the presequencewhich codes for the signal peptide of Bacillus pumilus PRL B12 xylanase.The invention also relates to vectors which contain this promoter andthis presequence, and also to the DNA molecule comprising the nucleotidesequence which codes for the mature portion of the xylanase of theinvention. The invention also relates to strains transformed by thesevectors.

[0004] Thermostable xyalanases which are active over a wide pH range areknown, such as, in particular, xylanases produced by strains ofalkalophilic bacillus (Gupta et al. Biotechnology Letters, 1992, 14(11), pages 1045-1046 and International Patent Application WO 94/04664).However, despite these properties, these enzymes would appear to bepoorly effective in bleaching paper pulp.

[0005] Consequently, there is at present a need for a xylanase which canbe used in the treatment of paper pulp, which is very stable and alsovery active over a wide range of temperature and of basic and acid pH.

[0006] The object of the present invention is to provide a new xylanasewhich is active over a wide pH range, both at alkaline pH and at acidpH.

[0007] The object of the present invention is also to identify, isolateand provide a strain, especially a Bacillus strain, which produces thesaid xylanase naturally.

[0008] The object of the present invention is also to isolate andprovide a DNA molecule comprising a nucleotide sequence which codes forthe said xylanase.

[0009] The object of the present invention is also to prepare andprovide an expression vector containing the nucleotide sequence codingfor the said xylanase.

[0010] The object of the present invention is also to prepare andprovide an integration vector containing the nucleotide sequence codingfor the said xylanase.

[0011] The object of the present invention is also to prepare andprovide the promoter drived from the gene which codes for Bacilluspumilus PRL B12 xylanase. The object of the present invention is also toprepare and provide the presequence which codes for the signal peptideof Bacillus pumilus PRL B12 xylanase. The vectors which comprise thispromoter and/or this presequence also contain the DNA moleculecomprising the nucleotide sequence which codes for the mature portion ofthe xylanase of the invention. The strains transformed by these vectorsproduce the xylanase of the invention heterologously.

[0012] The object of the present invention is also to prepare andprovide a Bacillus host transformed with the expression vector whichcontains the DNA molecule comprising the nucleotide sequence of theBacillus strain coding for the said xylanase.

[0013] The object of the present invention is also to prepare andprovide a Bacillus host transformed with the expression vector whichcontains the DNA molecule comprising the nucleotide sequence of theBacillus strain coding for the said xylanase [sic].

[0014] The object of the present invention is also to prepare andprovide a composition containing this xylanase.

[0015] The object of the present invention is also to prepare andprovide a xylanase which can be used in the treatment of paper pulp, andpulps having a basic, neutral or acid pH, and in particular pulps havingan especially basic pH and paper pulps of various origins, such as thepulps originating from coniferous trees, the pulps originating frombroad-leaved trees and especially eucalyptus pulp.

[0016] To this end, the invention relates to a xylanase originating froma Bacillus, and more especially from an aerobic and non-thermophilicmicroorganism.

[0017] It is preferable to use Bacillus sp. strain 720/1 or a derivativeor mutant of this strain. The xylanase of the invention is derived from(naturally produced by) Bacillus sp. strain 720/1. Xylanase isclassified in the international system under the EC number 3.2.1.8. Itis an endo-1,4-beta-xylanase.

[0018] Preferably, the isolated and purified xylanase consists of asingle type of polypeptide having a molecular weight of approximately 25kDa.

[0019] The invention relates to an isolated and purified xylanasecomprising the amino acid sequence from 1 to 221 amino acids (SEQ IDNO:3) or a modified sequence derived from this sequence. The amino acidsequence and the nucleotide sequence (SEQ ID NO:1) coding for the maturexylanase, together with its translation into amino acids (SEQ ID NO:2),is given in FIG. 1 (FIGS. 1a and 1 b).

[0020] The xylanase of the invention is synthesized in the form of aprecursor. The precursor contains 248 amino acids: (SEQ ID NO:6). Thenucleotide sequence SEQ ID NO:4) coding for the xylanase precursor, aswell as its translation into amino acids (SEQ ID NO:5), are identified.

[0021] The precursor contains the sequence of 221 amino acids (SEQ IDNO:3) of the mature xylanase and the sequence of 27 amino acids (SEQ IDNO:9) of the presequence.

[0022] The mature xylanase sequence is preceded by a presequence. Thelatter is an additional sequence of 27 amino acids (SEQ ID NO:9). Thecorresponding nucleotide sequence (SEQ ID NO:7), as well as itstranslation into amino acids (SEQ ID NO:8), are identified. Thispresequence codes for the signal peptide of the xylanase of theinvention.

[0023] As a special preference, the said xylanase has a determinedisoelectric point of between approximately 9.5 and approximately 9.7.

[0024] The xylanase according to the invention is thermostable andactive over a wide pH range. Preferably, the xylanase according to theinvention is alkaline.

[0025] The xylanase according to the invention possesses, moreover, allappropriate properties compatible with the actual industrial conditionsof enzyme treatment of paper pulp. According to the numerous steps ofthe various treatments of paper pulp employed industrially, theseproperties are good stability with respect to pH and temperature, andenzyme activity over a wide range of pH and temperature, such as, inparticular, a pH of between approximately 5 and 10 and a temperature ofbetween approximately 50 and 80° C.

[0026] The xylanase of the invention is active over a range of pH aboveor equal to approximately 5. The xylanase of the invention is activeover a range of pH lower or equal to approximately 11. The xylanasedevelops an enzyme activity of more than 50% of the maximal activity,measured at a temperature of approximately 50° C. and in the presence ofxylan, over a range of pH above or equal to approximately 5.0. Thexylanase develops an enzyme activity of more than 50% of the maximalactivity, measured at a temperature of approximately 50° C. and in thepresence of xylan, over a pH range below approximately 10.5.

[0027] The xylanase of the invention is active over a range oftemperature above or equal to approximately 50° C. The xylanase of theinvention is active over a range of temperature below or equal toapproximately 80° C. The xylanase develops an enzyme activity of morethan 50% of the maximal activity, measured at a pH of approximately 9and in the presence of xylan, over a range of temperature above or equalto approximately 50° C. The xylanase develops an enzyme activity of morethan 50% of the maximal activity, measured at a pH of approximately 9and in the presence of xylan, over a temperature range belowapproximately 80° C.

[0028] The invention also relates to a modified xylanase, that is to sayan enzyme whose amino acid sequence differs from that of the wild-typeenzyme by at least one amino acid. These modifications may be obtainedby standard mutagenesis techniques on the DNA, such as exposure toultraviolet radiation or to chemical products such as ethylmethanesulphonate (EMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG),sodium nitrite or O-methyl-hydroxylamine, or by genetic engineeringtechniques such as, for example, site-directed mutagenesis or randommutagenesis. These techniques are known to a person skilled in the artand are described, in particular, in Molecular Cloning—a laboratorymanual—Sambrook, Fritsch, Maniatis—second edition, 1989, Chapter 15.

[0029] The invention also relates to a xylanase having immunochemicalproperties identical or partially identical to the xylanase obtainedfrom Bacillus sp. strain 720/1. The immunochemical properites may bedetermined immunologically by tests of identity, in particular usingspecific polyclonal or monoclonal antibodies. Tests of identity areknown to a person skilled in the art, such as, in particular, theOuchterlony immunodiffusion method or the immunoelectrophoresis method.Examples of such methods are described by Axelsen N. H., Handbook ofImmunoprecipitation Gel Techniques, Blackwell Scientific Publications,1983, Chapters 5 and 14; the terms “antigenic identity” and “partialantigenic identity” are described in this document in Chapters 5, 19 and20. A serum containing the specific antibody is prepared according tothe method described, by immunizing animals (for example mice, rabbitsor goats) with a purified xylanase preparation. This preparation may bemixed with an additive such as Freund's adjuvant, and the mixtureobtained is injected into animals. The polyclonal antibody is obtainedafter one or several immunizations. An example consists in injectingsubcutaneously at two-week intervals four fractions each containing 150micrograms of purified xylanase; the immunization then lasts 8 weeks.The serum is withdrawn after the immunization period and theimmunoglobulin may be isolated according to the method described byAxelsen N. H. (1983).

[0030] The present invention also relates to the identification andprovision of a new, isolated and purified aerobic bacterium producingxylanase. Generally, it belongs to the family Bacillaceae. Preferably,it belongs to the genus Bacillus. As a special preference, the saidBacillus is Bacillus sp. strain 720/1 or a derivative or mutant of thisstrain.

[0031] Derivative of this strain is understood to mean any naturallymodified bacterium. The derivatives of this strain may be obtained byknown modification techniques such as culture on specific medium,ultraviolet radiation or X-rays. Mutant of this strain is understood tomean any artificially modified bacterium. The mutants of this strain maybe obtained by known modification techniques such as exposure tomutagenic agents and genetic engineering techniques. These techniquesare known to a person skilled in the art and are described, inparticular, in Sambrook et al., 1989, Chapter 15.

[0032] Bacillus sp. strain 720/1 was deposited at the collection namedBelgian Coordinated Collections of Microorganisms (LMG culturecollection, Ghent University, Microbiology Laboratory—K. L.Ledeganckstraat 35, B-9000 Ghent, Belgium) in accordance with theBudapest Treaty under the number LMG P-14798 on Jun. 9, 1994. Theinvention relates to an isolated and purified culture of Bacillus sp.strain 720/1 and to a derived or mutated culture of the latter.

[0033] The strain of the present invention was identified by itsbiochemical features: aerobic Gram-positive bacterium which takes theform of a rod; it forms an endospore. It is oligosporogenous.

[0034] The invention also relates to the isolation and provision of aDNA molecule comprising the nucleotide sequence (SEQ ID NO:1) whichcodes for the mature xylanase of Bacillus sp. 720/1 (LMG P-14798) or amodified sequence derived from this sequence. Preferably, this DNAmolecule comprises the entire Bacillus sp. 720/1 xylanase gene. Entirexylanase gene (SEQ ID NO:10) is understood to mean at least thetranscription promoter(s), the signal sequence(s), the nucleotidesequence coding for the mature xylanase and the transcriptionterminator(s).

[0035] Modified sequence derived from the DNA molecule is understood tomean any DNA molecule obtained by modification of one or morenucleotides of the gene which codes for the xylanase of the invention.The techniques of obtaining such sequences are known to a person skilledin the art, and are described, in particular, in Molecular Cloning—alaboratory manual—Sambrook, Fritsch, Maniatis—second edition, 1989,Chapter 15. Usually, the modified sequence derived from the DNA moleculecomprises at least 70% homology with the nucleotide sequences [sic] (SEQID NO:1) of the gene which codes for the xylanase of the invention, thatis to say at least 70% of identical nucleotides having the same positionin the sequence. Preferably, the modified sequence derived from the DNAmolecule comprises at least 80% homology with the nucleotide sequence(SEQ ID NO:1) of the gene which codes for the xylanase of the invention.As a special preference, the modified sequence derived from the DNAmolecule comprises at least 90% homology with the nucleotide sequences[sic] (SEQ ID NO:1) of the gene which codes for the xylanase of theinvention.

[0036] The complete nucleotide sequence coding for the mature xylanase,together with its translation into amino acids (SEQ ID NO:2), is givenin FIG. 1 (FIGS. 1a and 1 b).

[0037] Usually, the DNA molecule according to the invention comprises atleast the nucleotide sequence (SEQ ID NO:4) which codes for the xylanaseprecursor or a modified sequence derived from this sequence. Thisnucleotide sequence (SEQ ID NO:4) comprises the nucleotide sequence (SEQID NO:1) coding for the mature xylanase of Bacillus sp. 720/1 (LMGP-14798) and its signal sequence (presequence) (SEQ ID NO:7).Preferably, this DNA molecule comprises the entire Bacillus sp. 720/1xylanase gene and, as a special preference, the nucleotide sequence (SEQID NO:10). The nucleotide sequence (SEQ ID NO:10) consists, in theamino-carboxy direction and from left to right, of the nucleotidesequence (SEQ ID NO:12) which comprises the xylanase promoter, thenucleotide sequence of the presequence (SEQ ID NO:7), the nucleotidesequence of the mature xylanase (SEQ ID NO:1) and the nucleotidesequence (SEQ ID NO:13) which comprises the xylanase terminator. FIG. 2(FIG. 2a and FIG. 2b) shows the nucleotide sequence of the gene codingfor the xylanase, together with its translation into amino acids (SEQ IDNO:11).

[0038] In a variant, the invention also relates to a DNA molecule whichcomprises the promoter derived from the gene which codes for Bacilluspumilus PRL B12 xylanase, a presequence and the nucleotide sequence (SEQID NO:1) which codes for Bacillus sp. 720/1 xylanase or a modifiedsequence derived from this sequence. In another variant, the inventionalso relates to a DNA molecule which comprises a promoter, thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase and the nucleotide sequence (SEQ ID NO:1) which codes forBacillus sp. 720/1 xylanase or a modified sequence derived from thissequence. Preferably, the invention relates to a DNA molecule whichcomprises the promoter (SEQ ID NO:26) derived from the gene which codesfor Bacillus pumilus PRL B12 xylanase, the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanaseand the nucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence.

[0039] The invention also relates to the promoter (SEQ ID NO:26) derivedfrom the gene which codes for Bacillus pumilus PRL B12 xylanase. Thesequence of the promoter is illustrated in FIG. 11.

[0040] The invention also relates to the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanase.The corresponding sequence of 27 amino acids has been identified (SEQ IDNO:29). This nucleotide sequence, together with its translation intoamino acids (SEQ ID NO:28), is illustrated in FIG. 12.

[0041] The method for obtaining and preparing the promoter derived fromthe gene which codes for Bacillus pumilus PRL B12 xylanase and of thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase is described in Example 17 and in FIG. 1 of European PatentApplication 0,634,490, which is incorporated by reference in thisapplication.

[0042]Bacillus pumilus strain PRL B12 was deposited at the ATCCcollection (American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., 20852, USA) in accordance with the Budapest Treaty underthe number ATCC 55443 on Jun. 24, 1993.

[0043] The invention also relates to a mutated DNA molecule, and to themutated xylanase derived therefrom (for which the mutated DNA moleculecodes), obtained by modification of the nucleotide sequence of the genewhich codes for the xylanase defined above. The techniques of obtainingsuch mutated xylanases are known to a person skilled in the art and aredescribed, in particular, in Molecular Cloning—a laboratorymanual—Sambrook, Fritsch, Maniatis—second edition, 1989, Chapter 15.

[0044] The present invention also relates to an expression vector orchromosomal integration vector containing a DNA molecule as definedabove. Generally, the expression vector or the chromosomal integrationvector contains the DNA molecule which comprises the nucleotide sequence(SEQ ID NO:1) which codes for Bacillus sp. 720/1 xylanase or a modifiedsequence derived from this sequence. Usually, the expression vector orthe chromosomal integration vector contains a DNA molecule whichcomprises the gene which codes for the xylanase or a modified sequencederived from this sequence. Preferably, the expression vector or thechromosomal integration vector contains the DNA molecule which comprisesthe nucleotide sequence (SEQ ID NO:10) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence. As aspecial preference, this vector is the expression vector pUBRD-720X11.Good results have also been obtained with the expression vectorpUBR-720X11.

[0045] A variant of the invention relates to an expression vector or achromosomal integration vector which contains a DNA molecule comprisingthe gene which codes for the mature portion of the xylanase or amodified sequence derived from this molecule. Generally, the expressionvector or the chromosomal integration vector contains the DNA moleculewhich comprises the nucleotide sequence (SEQ ID NO:1) which codes forBacillus sp. 720/1 xylanase or a modified sequence derived from thissequence. Usually, the expression vector or the chromosomal integrationvector contains a DNA molecule which comprises the promoter derived fromthe gene (SEQ ID NO:26) which codes for Bacillus pumilus PRL B12xylanase, a presequence and the nucleotide sequence (SEQ ID NO:1) whichcodes for Bacillus sp. 720/1 xylanase or a modified sequence derivedfrom this sequence. In a usual variant, the expression vector contains aDNA molecule which comprises a promoter, the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanaseand the nucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence.Preferably, the expression vector or the chromosome integration vectorcontains a DNA molecule which comprises the promoter derived from thegene (SEQ ID NO:26) which codes for Bacillus pumilus PRL B12 xylanase,the presequence (SEQ ID NO:27) which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase and the nucleotide sequence (SEQ IDNO:1) which codes for Bacillus sp. 720/1 xylanase or a modified sequencederived from this sequence. As a special preference, this vector is theexpression vector pBPXD-PRE-720X. Good results have also been obtainedwith the expression vector pC-BPX-PRE-720X.

[0046] The invention also relates to an expression system which can beused for the production of a polypeptide.

[0047] This expression system comprises:

[0048] the sequence of the promoter (SEQ ID NO:26) derived from the genewhich codes for Bacillus pumilus PRL B12 xylanase,

[0049] a sequence coding for a signal peptide, and

[0050] the sequence of the polypeptide of interest.

[0051] Generally, the expression system comprises the sequence of aterminator.

[0052] In a variant, this expression system comprises:

[0053] the sequence of a promoter,

[0054] the presequence (SEQ ID NO:27) which codes for the signal peptideof Bacillus pumilus PRL B12 xylanase, and

[0055] the sequence of the polypeptide of interest.

[0056] Generally, the expression system comprises the sequence of aterminator.

[0057] Usually, this expression system comprises:

[0058] the sequence of the promoter (SEQ ID NO:26) derived from the genewhich codes for Bacillus pumilus PRL B12 xylanase,

[0059] the presequence (SEQ ID NO:27) which codes for the signal peptideof Bacillus pumilus PRL B12 xylanase,

[0060] the sequence of the polypeptide of interest, and

[0061] the sequence of a terminator.

[0062] Preferably, the polypeptide of interest is an enzyme such as ahydrolase. As a special preference, the polypeptide of interest is aprotease, a lipase, a xylanase, a cellulase, an amylase or apullulanase. Good results have been obtained with the xylanase naturallyproduced by Bacillus sp. strain 720/1, that is to say when, in theexpression system, the sequence of the polypeptide corresponds to thenucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp. 720/1xylanase.

[0063] The present invention also relates to recombinant strains intowhich the gene coding for a xylanase is introduced by geneticengineering techniques. The gene may be introduced by means of areplicative vector, or integrated in the host's chromosome in one ormore copies by means of an integrative vector; the nucleotide sequencecoding for a xylanase may be introduced by transformation, either inintegrated form in the chromosomal DNA, or in self-replicating form(plasmid).

[0064] The invention also relates to strains of microorganisms organismswhich are different from the initial producer organism, into whichstrains the nucleotide sequence coding for a xylanase is introduced bytransformation, either in integrated form in the chromosomal DNA, or inself-replicating form (plasmid); the gene coding for a xylanase may beintroduced by means of a replicative vector or integrated in the host'schromosome in one or more copies by an integrative vector.

[0065] The invention relates to a transformed strain comprising the DNAmolecule which contains the structural gene which codes for the maturexylanase of Bacillus sp. 720/1. Generally, the transformed strain is astrain of bacterium. Usually, the transformed strain is chosen fromEscherichia, Pseudomonas or Bacillus strains. Preferably, thetransformed strain is a Bacillus strain. As a special preference, thetransformed Bacillus strain is a Bacillus licheniformis strain, aBacillus pumilus strain, a Bacillus alcalophilus strain or a Bacillussp. strain 720/1. Good results have been obtained with a Bacilluslicheniformis strain and with a Bacillus pumilus strain.

[0066] The invention relates to the transformed Bacillus straincomprising the expression vector or the chromosomal integration vectorwhich comprises this DNA molecule. Preferably, the transformed Bacillusstrain is a Bacillus licheniformis strain. Preferably also, thetransformed Bacillus strain is a Bacillus sp. strain 720/1.

[0067] The present invention also relates to the xylanase produced by atransformed strain as defined above.

[0068] The present invention also relates to a method for the productionof a xylanase, comprising the culturing of an aerobic bacterium capableof producing the xylanase in a suitable nutrient medium containingcarbon and nitrogen sources and inorganic salts under aerobicconditions, and the harvesting of the xylanase thereby obtained. Thisculture medium can be solid or liquid. Preferably, the culture medium isliquid. Preferably, the aerobic bacterium is a Bacillus strain or aderivative of this strain capable of producing the xylanase.

[0069] The present invention also relates to a method for the productionof a xylanase, comprising the culturing of Bacillus sp. strain 720/1 ora derivative of this strain capable of producing the xylanase in asuitable nutrient medium containing carbon and nitrogen sources andinorganic salts under aerobic conditions, and the harvesting of thexylanase thereby obtained.

[0070] The invention also relates to a method for the preparation of axylanase from a recombinant organism, the method comprising theisolation of a DNA fragment coding for the xylanase, the insertion ofthis DNA fragment into a suitable vector, the introduction of thisvector into a suitable host or the introduction of this DNA fragmentinto the chromosome of a suitable host, the culturing of this host, theexpression of the xylanase and the harvesting of the xylanase. Thesuitable host is generally chosen from the group consisting ofEscherichia coli, Bacillus or Aspergillus microorganisms. Usually, thehost is chosen from Bacillus species. Preferably, the host is chosenfrom microorganisms of the genus Bacillus (aerobic). As a specialpreference, the host is chosen from the microorganisms Bacillussubtilis, Bacillus licheniformis, Bacillus alcalophilus, Bacilluspumilus, Bacillus lentus, Bacillus amyloliquefaciens or Bacillus sp.720/1. Good results have been obtained when the host for the expressionof the xylanase according to the present invention is a recombinantstrain derived from Bacillus licheniformis, and preferably Bacilluslicheniformis strain SE2 delap1 and Bacillus licheniformis strain SE2delap6. Bacillus licheniformis strain SE2 delap1 and Bacilluslicheniformis strain SE2 delap6 are described in European PatentApplication 0,634,490, which is incorporated by reference in thisapplication.

[0071] The invention also relates to a xylanase produced heterologouslyby a microorganism of the genus Bacillus. Usually, the microorganism ofthe genus Bacillus contains a gene coding for an alkaline protease whenit is in the wild-type state. Preferably, this microorganism is aBacillus licheniformis strain comprising the DNA molecule whichcomprises the nucleotide sequence which codes for Bacillus sp. 720/1xylanase. As a special preference, the gene coding for the alkalineprotease has been removed by deletion from this Bacillus strain. Thisstrain is preferably Bacillus licheniformis strain SE2 delap1 orBacillus licheniformis strain SE2 delap6.

[0072] Produced heterologously is understood to mean a production whichis not performed by the natural microorganism, that is to say themicroorganism which, in the wild-type state, contains the gene whichcodes for the xylanase.

[0073] The conditions of culture of these bacteria, such as componentsof the nutrient medium, culture parameters, temperature, pH, aerationand agitation, are well known to a person skilled in the art. Examplesof such techniques are described, in particular, in Ullmann'sEncyclopedia of Industrial Chemistry, 1987, 5th Edition, Vol. A9, pages363-390.

[0074] The techniques of harvesting of xylanase are well known to aperson skilled in the art, and are chosen according to the usesenvisaged for the xylanase. Usually, centrifugation, filtration,ultrafiltration, evaporation, microfiltration, crystallization or acombination of one or other of these techniques is used, such as acentrifugation followed by an ultrafiltration. Examples of suchtechniques are described, in particular, by R. Scriban, Biotechnology,(Technique et Documentation Lavoisier), 1982, pp. 267-276 and inUllmann's Encyclopedia of Industrial Chemistry, 1987, 5th Edition, Vol.A9, pages 363-390.

[0075] The xylanase can then be purified, if necessary and according tothe uses envisaged. Enzyme purification techniques are well known to aperson skilled in the art, such as precipitation using a salt such asammonium sulphate, or using a solvent such as acetone or an alcohol.Examples of such techniques are described, in particular, by R. Scriban,Biotechnology, (Technique et Documentation Lavoisier), 1982, pp.267-276.

[0076] The xylanase may also be dried by atomization or lyophilization.Examples of such techniques are described, in particular, by R. Scriban,Biotechnology, (Technique et Documentation Lavoisier), 1982, pp. 267-276and in Ullmann's Encyclopedia of Industrial Chemistry, 1987, 5thEdition, Vol. A9, pages 363-390.

[0077] The present invention also relates to enzyme compositionscomprising the xylanase according to the invention and at least oneadditive. These additives are known to a person skilled in the art andare chosen according to the use envisaged for the composition. They mustbe compatible with the xylanase and must have little or no effect on theenzyme activity of the xylanase. Usually, these additives are enzymestabilizers, preservatives and formulation agents.

[0078] The compositions comprising the xylanase of the present inventionmay be used in solid or liquid form.

[0079] The xylanase is formulated according to the anticipated uses.Stabilizers or preservatives may also be added to the enzymecompositions comprising the xylanase according to the invention. Forexample, it is possible to stabilize the xylanase by adding propyleneglycol, ethylene glycol, glycerol, starch, xylan, a sugar such asglucose and sorbitol, a salt such as sodium chloride, calcium chloride,potassium sorbate and sodium benzoate or a mixture of two or more ofthese products. Good results have been obtained with propylene glycol.Good results have been obtained with sorbitol.

[0080] The xylanase according to the invention has numerous outlets invarious industries such as, for example, the food industries, thepharmaceutical industries or the chemical industries.

[0081] The xylanase may be used, in particular, in bakery. An example ofuse of a xylanase in bakery is described, in particular, inInternational Patent Application WO 94/04664.

[0082] The xylanase can be used, in particular, for the treatment ofpaper pulp. An example of the use of a xylanase for the treatment ofpaper pulp is described, in particular, in European Patent Application0,634,490. The xylanase of the present invention is effective, inparticular, on the pulp originating from eucalyptus wood, as illustratedin Example 13 of the present patent application.

[0083] The xylanase can be used, in particular, in animal feeds. Anexample of a use of a xylanasse in animal feeds is described, inparticular, in European Patent Application 0,507,723.

[0084]FIG. 1 (FIG. 1a and FIG. 1b) shows the nucleotide sequence (SEQ IDNO:2) coding for the mature xylanase, together with its translation intoamino acids.

[0085]FIG. 2 (FIG. 2a and FIG. 2b) shows the nucleotide sequence (SEQ IDNO:11) of the gene coding for xylanase, together with its translationinto amino acids.

[0086]FIG. 3 shows the restriction map of plasmid pUBR2002.

[0087]FIG. 4 shows the restriction map of plasmid pUBR-720X1.

[0088]FIG. 5 shows the restriction map of plasmid pUBR-720X11.

[0089]FIG. 6 shows the restriction map of plasmid pUBRD-720X11.

[0090]FIG. 7 shows the restriction map of plasmid pUBC2001.

[0091]FIG. 8 shows the restriction map of plasmid pC-BPX-PRE-2003

[0092]FIG. 9 shows the restriction map of plasmid pC-BPX-PRE-720X.

[0093]FIG. 10 shows the restriction map of plasmid pBPXD-PRE-720X.

[0094]FIG. 11 shows the promoter (SEQ ID NO:26) derived from the genewhich codes for Bacillus pumilus PRL B12 xylanase.

[0095]FIG. 12 shows the presequence (SEQ ID NO:28) which codes for thesignal peptide of Bacillus pumilus PRL B12 xylanase.

[0096] The meaning of the abbreviations and symbols used in thesefigures is collated in the following table. Symbol Abbreviation MeaningOriEC origin of replication in E. coli REP protein needed forreplication in Bacillus Ori+ origin of replication of the + strand inBacillus Ori− origin of replication of the − strand in Bacillus AmpRgene conferring resistance to ampicillin KmR gene conferring resistanceto kanamycin BlmR gene conferring resistance to bleomycin 5′720XYL 5′sequence located upstream of the sequence coding for Bacillus sp. 720/1xylanase 3′720XYL 3′ sequence located downstream of the sequence codingfor Bacillus sp. 720/1 xylanase 720XYL sequence coding for the Bacillussp. 720/1 xylanase precursor 5′BPUXYL-PRE promoter and ribosome bindingsite of Bacillus pumilus PRL B12 xylanase, followed by the presequenceof Bacillus pumilus PRL B12 xylanase 720XYL-NAT sequence coding for themature portion of Bacillus sp. 720/1 xylanase

[0097] The present invention is illustrated by the examples whichfollow.

EXAMPLE 1

[0098] Isolation and Characterization of Bacillus sp. Strain 720/1

[0099] Bacillus sp. strain 720/1 was isolated from a sample of soil,obtained in Argentina, on a nutrient agar medium, and selected for itscapacity to degrade a coloured xylan derivative known by the name ofAZCL-xylan and sold by the company Megazyme.

[0100] This strain was cultured at 37° C. in LBS/C growth medium whosecomposition is as follows: wheat bran 10 g/l, Tryptone (Difco) 10 g/l,yeast extract 5 g/l, NaCl 10 g/l, Na₂CO₃ 5.3 g/l, NaHCO₃ 4.2 g/l.

[0101] The sodium carbonate and bicarbonate are sterilized separatelyand then added aseptically to the other components of the sterilemedium. The agar medium contains, in addition, 20 g/l of agar. Thestrain of the present invention was identified by its biochemicalfeatures: aerobic Gram positive bacterium which takes the form of a rod;it forms an endospore. Hence it belongs to the genus Bacillus.

[0102] The vegetative cells of this strain in culture on LBS/C agarmedium at 37° C. have a bacillus shape 0.8×3.0-5 μm in size. Themobility of the vegetative cells is positive.

[0103] After growth for 13 days at 37° C. on TSA agar medium,microscopic observation reveals the presence of sporangia. TSA agarmedium contains 15 g/l of Tryptone (Difco), 5 g/l of soya bean peptone,5 g/l of NaCl and 15 g/l of agar.

[0104] The strain is oligosporogenous.

[0105] The test for catalase is positive in the presence of 10% (v/v) ofhydrogen peroxide. The test for oxydase is positive in the presence of1% (w/v) of tetramethyl-1,4-phenylenediammonium dichloride.

[0106] This strain is aerobic, that is to say grows under aerobicconditions. It does not grow under anaerobic conditions, that is to sayunder an atmosphere of 84% (v/v) N₂, 8% (v/v) CO₂, 8% (v/v) H₂ at 37° C.The abbreviation % (v/v) represents a percentage expressed in terms ofvolume per volume.

[0107] This strain is not thermophilic. It displays normal growth afterincubation in LBS/C agar medium at 20° C., 30° C, 37° C. and 45° C.; incontrast, it does not grow at 50° C. and 55° C., or at 10° C.

[0108] It displays normal growth after incubation in LBS/C agar mediumin the presence of NaCl at concentrations of 2.0% (w/v) and 3.5% (w/v),and displays slight growth in the presence of 5.0% (w/v) and 7.0% (w/v)NaCl. The abbreviation % (w/v) represents a percentage expressed interms of weight per volume.

[0109] Bacillus sp. strain 720/1 does not acifify glucose.

[0110] Bacillus sp. strain 720/1 has been identified by means of the API50 CHB strip and the API 20 E strip following the instructions for useof the supplier (API System, France). Bacillus sp. strain 720/1 utilisesglycerol, N-acetylglucosamine, arbutin, citrate, galactose, amygdalinand melibiose, and hydrolyses gelatin. These features differentiateBacillus sp. strain 720/1 clearly from a Bacillus pumilus strain. Ineffect, a Bacillus pumilus strain does not display any of thesefeatures.

[0111] Bacillus sp. strain 720/1 was also identified by means of theBiolog system (USA). The data bank analysing the results of this systemgives a score of 0.564 for Bacillus coagulans, 0.097 for Bacillussubtilis, 0.057 for Bacillus licheniformis and 0.00 for Bacilluspumilus. These features differentiate Bacillus sp. strain 720/1 clearlyfrom a Bacillus coagulans strain, a Bacillus subtilis strain, from aBacillus licheniformis strain and from a Bacillus pumilus strain.

[0112] Hence the isolated bacterium belongs to the genus Bacillus; noknown species could be determined.

[0113] Bacillus sp. strain 720/1 was deposited at the collection namedBelgian Coordinated Collections of Microorganisms (LMG culturecollection) under the number LMG P-14798.

EXAMPLE 2

[0114] Production of Xylanase by Bacillus sp. 720/1

[0115] Bacillus sp. strain 720/1 is cultured on Petri dishes containingan LBS/C agar medium at 37° C. for 48 hours (culture A).

[0116] Then, from the culture A, culturing is carried out in an LB/Cliquid medium whose composition is identical to that of LBS/C medium butwithout wheat bran, at 37° C. for 24 hours with orbital shaking at therate of 250 rpm (culture B) with an amplitude of approximately 2.54 cm.

[0117] 500 ml of the culture B are then transferred to a 20-1 fermentercontaining 14 1 of LBS/C medium. The pH is allowed to find its naturalvalue, and the speed of agitation and the flow rate of air blown intothe fermenter are such that the partial pressure of oxygen dissolved inthe culture medium is not below 30% of the saturation value.

[0118] After 72 hours of culture at 37° C., the xylanase and thecellular biomass are separated by centrifugation (Beckman J21, JA10rotor) at 8,000 rpm for 30 minutes. The xylanase produced by Bacillussp. strain 720/1 is extracellular. From the centrifugation supernatant,the residual insoluble matter is then separated from the xylanase bymicrofiltration (KROS FLOII cartridge, porosity 0.2 μ, companyMicrogon).

[0119] The microfiltration retentate is washed with 1 1 of demineralizedwater. This washing is performed three times.

[0120] The permeate of this microfiltration is then concentratedapproximately 20-fold by ultrafiltration through a Pall MICROZA SIP 1013polysulphone cartridge having a cut-off threshold of 6 kD (companyPall).

[0121] The enzyme activity is measured on the ultrafiltration retentate(product R) and permeate (product P) obtained.

[0122] One xylanase enzyme unit (IU) is defined as the amount of enzymewhich, at pH 8.0, at a temperature of 50° C. and in the presence ofxylan, catalyses the liberation of glucose equivalents at the rate of 1μmol of glucose per minute (μM [sic]/minute).

[0123] The measurement of xylanase enzyme activity is carried outaccording to the protocol described by Bailey, Biely and Poutanen, J.Biotechnology, 1992, 23, pages 257-270; except that thecitrate-phosphate buffer mentioned by Bailey et al. was replaced by 50mM tris(hydroxymethyl)aminoethane-HCl buffer (pH 8.0).

[0124] Sufficient polyethylene glycol (Merck polyethylene glycolreference 807490) is added to the ultrafiltration retentate (product R)to obtain a concentration of 40% (w/w). After solubilization of thepolyethylene glycol, the solution obtained is incubated for 30 minutesat 25° C.

[0125] The solution containing the polyethylene glycol and the xylanaseis then centrifuged for 10 minutes at 8,000 rpm (Beckman J21 centrifuge,JA10 rotor). The supernatant is removed by centrifugation. SufficientNaCl solution (0.9% v/v) is added to the centrifugation pellet torecover the initial volume of the retentate used (product R).

[0126] Sufficient acetone is then added to this suspension containingthe xylanase and NaCl to achieve a concentration of 40% (v/v). Thisacetone suspension is incubated for 45 minutes at 4° C.

[0127] After this incubation, this acetone suspension is centrifuged for10 minutes at 8,000 rpm (Beckman J21, JA10 rotor).

[0128] The centrifugation supernatant is retained. To thiscentrifugation supernatant, acetone is added to a concentration of 80%(v/v). This acetone suspension is incubated for 45 minutes at 4° C.

[0129] After this incubation, this acetone suspension is centrifuged for10 minutes at 8,000 rpm (Beckman J21, JA10 rotor).

[0130] The centrifugation pellet is retained. It is suspended in asufficient volume of 0.9% (v/v) NaCl solution to be solubilized (productN).

EXAMPLE 3

[0131] Purification of the Xylanase

[0132] A fraction of the ultrafiltration retentate (product N) obtainedin Example 2 is conditioned by passage through a gel permeationchromatography column (Bio-Rad Econopac 10DG column) equilibrated with20 mM Bis-Tris(bis(2-hydroxyethyl)iminotris(hydroxy-methyl)methane)buffer, pH 6.2 (buffer A). A solution designated product X is therebyobtained.

[0133] 1 ml of the product X solution is then applied to an S SepharoseHP 16/10 (Pharmacia) cation exchange column previously equilibrated withthe buffer A. The flow rate is 2.5 ml per minute, with an isocraticelution for 10 minutes, followed by an NaCl concentration gradient (from10 to 50 minutes; the NaCl content rises from 0 to 0.7 M). A single peakis detected during the gradient, corresponding to the elution of thexylanase.

[0134] The fractions containing the xylanase activity (solution A) arecollected.

[0135] It is verified that these fractions contain xylanase by applying10 μl of each fraction to an agar medium comprising xylan (mediumcontaining 0.5 g/l of AZCL-xylan, 50 mM Tris buffer (pH 8.0) and 15 g/lof agar). A halo forms around the fractions which contain xylanase.

EXAMPLE 4

[0136] Amino Acid Sequence

[0137] The amino acid sequence of the xylanase of the present inventionis determined indirectly from the nucleotide sequence (SEQ ID NO:10) ofthe gene which codes for this xylanase, which is obtained as describedin Example 14. This is carried out using the IntelliGenetics SuiteSoftware for Molecular Biology (Release #5.4) computer program ofIntelliGenetics, Inc. USA. FIG. 2 (FIG. 2a and FIG. 2b) shows thenucleotide sequence (SEQ ID NO:10) of the gene coding for the xylanase,together with its translation into amino acids (SEQ ID NO:11).

[0138] The xylanase is synthesized in the form of a precursor. Thexylanase precursor contains 248 amino acids (SEQ ID NO:6). Thenucleotide sequence (SEQ ID NO:4) coding for the xylanase precursor, aswell as its translation into amino acids (SEQ ID NO:5), are identified.

[0139] The presequence of the xylanase synthesized in the form of aprecursor is identified. It is a sequence of 27 amino acids (SEQ IDNO:9). The corresponding nucleotide sequence (SEQ ID NO:7) isidentified.

[0140] The amino acid sequence of the mature xylanase is thenidentified. The mature xylanase contains 221 amino acids (SEQ ID NO:3).

[0141]FIG. 1 (FIG. 1a and FIG. 1b) shows the nucleotide sequence (SEQ IDNO:1) coding for the mature xylanase, together with its translation intoamino acids (SEQ ID NO:2). cl EXAMPLE 5

[0142] Amino Acid Distribution

[0143] The amino acid distribution of the mature xylanase, determinedfrom the amino acid sequence (SEQ ID NO:3) of the xylanase (Example 4),is summarized in Table 1. TABLE 1 % (in molecular Symbol Amino acidNumber weight) N asparagine 25 11.6 Y tyrosine 13 8.6 T threonine 18 7.4S serine 19 6.7 I isoleucine 14 6.4 V valine 14 5.6 G glycine 24 5.5 Wtryptophan  7 5.3 K lysine 10 5.2 R arginine  8 5.1 Q glutamine  9 4.7 Daspartic acid 10 4.7 L leucine 10 4.6 E glutamic acid  8 4.2 Fphenylalanine  7 4.2 P proline  7 2.8 M methionine  5 2.7 A alanine  82.3 H histidine  4 2.2 C cysteine  1 0.4 B aspartic acid/asparagine  00.0 X unknown  0 0.0 Z glutamine glutamic acid  0 0.0

EXAMPLE 6

[0144] Estimation of the Molecular Weight

[0145] The molecular weight of the xylanase is estimated by calculationfrom the amino acid sequence of the mature form of the xylanase, asdescribed in Example 4.

[0146] A molecular weight of 24698.61 daltons is deduced by calculation.

EXAMPLE 7

[0147] Molecular Weight Determination

[0148] Concentration on a Centricon 10 kD device (Amicon) is performedon the solution A containing the xylanase, as obtained in Example 3.

[0149] 100 μl of the concentrated solution are applied to a Superdex 75HR 10/30 (Pharmacia) gel permeation chromatography column. The columnwas previously calibrated by means of the (Pharmacia) Gel Filtration LMWcalibration kit, code 17-0442-01, molecular weight markers. Elution tookplace at 0.25 ml/minute by means of 25 mM CAPSO(3-(cyclohexylamino)-2-hydroxy-1-propanesul-phonic acid) buffer pH 9.2,with the addition of 0.2 M NaCl.

[0150] The chromatogram obtained shows a single peak corresponding tothe xylanase activity. An apparent molecular weight of the protein ofapproximately 13.5 kD is deduced from this.

[0151] Polyacrylamide gel electrophoresis under denaturing conditions(SDS-PAGE) is also performed on the fraction originating from thissingle peak. The gel system used is the PhastSystem system of PharmaciaLKB Biotechnology, with gels containing a polyacrylamide gradient from10-15% (v/v). Electrophoresis conditions are those prescribed by thesupplier. Pharmacia LMW (Low Molecular Weight) molecular weight markers,reference 17-0446-01, are used as control. The markers employed arephosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carbonicanhydrase (30 kD), trypsin inhibitor (20.1 kD) and alpha-lactalbumin(14.4 kD).

[0152] Staining with Coomassie blue reveals a polypeptide of molecularweight approximately 25.7 kD.

EXAMPLE 8

[0153] Estimation of the Isoelectric Point

[0154] The isoelectric point of the xylanase is estimated from the aminoacid sequence of the mature form of the xylanase, as described inExample 4.

[0155] The estimated isoelectric point represents the net charge of theprotein in denatured form.

[0156] An isoelectric point of 7.46 is deduced for the xylanase indenatured form.

EXAMPLE 9

[0157] Isoelectric Point Determination

[0158] A fraction of the solution A, as obtained in Example 3, isapplied to a Mono P 5/20 chromatofocusing column (Pharmacia), followingthe supplier's recommendations, previously equilibrated with a 25 mMdiethanolamine buffer, pH 9.9.

[0159] The column is eluted by means of the Polybuffer 96 ampholytesolution (Pharmacia) diluted 10-fold in demineralized water.

[0160] The pH of the fraction containing the xylanase activity is 9.5.

[0161] It is verified that this fraction contains xylanase by applying10 μl of the fraction to an agar medium comprising xylan (mediumcontaining 0.5 g/l of AZCL-xylan, 50 mM Tris buffer (pH 8.0) and 15 g/lof agar). A halo, which takes the form of a zone of hydrolysis ofAZCL-xylan, forms around the fraction which contains the xylanase.

[0162] Isoelectric focusing is carried out on a fraction of the solutionA as obtained in Example 3.

[0163] To do this, Pharmacia DryIEF gel is rehydrated with a mixtureconsisting of 2 ml of demineralized water, 150 μl of Biolyte 8-10product (BioRad) and 75 μl of Pharmalyte 8-10.5 product (Pharmacia).Approximately 200 nanogrammes of proteins (fraction of the solution A)are applied to the gel. The protocol described by the supplier isfollowed.

[0164] It is deduced from this that the xylanase has an isoelectricpoint slightly above 9.6, the isoelectric point of the marker having thehighest isoelectric point used.

[0165] The experimentally observed isoelectric point represents thesurface charge of the protein in its native form.

EXAMPLE 10

[0166] Activity Profile as a Function of pH for the Xylanase Produced bythe Natural Strain (Bacillus sp. 720/1)

[0167] The enzyme activity of the xylanase is measured according to themethod described in Example 2 in the presence of xylan (Roth, reference7500, birchwood xylan) at a temperature of 50° C. and at different pHvalues (from 5.6 to 10.35) in different buffers chosen to obtain thedesired pH. The solution comprising the xylanase as obtained in Example2 (product N) is employed.

[0168] The results are collated in Table 2. It may be noted that themargin of error is estimated at approximately 25% in this type ofmeasurement.

[0169] In the course of this assay, maximal enzyme activity was measuredfor the sample placed at a pH of approximately 6.2 and at a temperatureof approximately 50° C. for 15 minutes. By definition, a relative enzymeactivity of 100% was hence assigned to this sample.

[0170] This example shows that the xylanase according to the inventiondevelops considerable enzyme activity over a pH range betweenapproximately 5.6 and approximately 10. TABLE 2 Relative Buffer usedactivity pH (50 mM) % 5.6 Tris-maleate  85 6.2 Tris-maleate 100 6.8Tris-maleate  96 7.5 Tris-  88 maleate/Tris* 8.7 Tris/Capso*  92 9.5Capso/Caps*  76 10 Caps  50 10.35 Caps  19

[0171] Tris=tris(hydroxymethyl)aminomethane

[0172] Tris-maleate=buffer composed of tris(hydroxy-methyl)aminomethane(50 mM) and maleic acid (50 mM), in which the ratio between thecomponents is chosen in accordance with the desired pH, the pH beingadjusted by means of NaOH (1 M)

[0173] Capso=3-(cyclohexylamino)-2-hydroxy-1-propanesulphonic acid

[0174] Caps=3-(cyclohexylamino)-1-propanesul-phonic acid

[0175] The symbol * means that the value obtained is the mean of themeasurements performed at the same pH but obtained with the two buffers.

[0176] This example shows that the xylanase according to the inventiondevelops high enzyme activity over a very wide pH range.

EXAMPLE 11

[0177] Effect of pH on the Activity of the Xylanase Produced by Bacillussp. Strain 720/1 as an Aid to the Bleaching of Coniferous Wood Pulp

[0178] Three aqueous suspensions of a pinewood pulp (obtained from thecompany SCA) are prepared, having a consistency of 2.5% (as weight ofdry matter) and having an initial Kappa number of 17.

[0179] The pH of these suspensions is adjusted to pH 5 with H₂SO₄ [sic].

[0180] 1st Stage: Enzyme Stage (Stage X)

[0181] The solution designated product N, as obtained in Example 2, isdiluted with demineralized water to obtain an enzyme solution having anenzyme activity of 25 IU/ml (as described in Example 2).

[0182] This enzyme solution containing the xylanase is added to onesuspension of pinewood pulp such that the pulp suspension is treated bymeans of 5 IU/g of dry pulp.

[0183] To the other two suspensions of pinewood pulp, demineralizedwater is added in place of the enzyme solution in identical proportions.

[0184] The three suspensions are then incubated for 2 hours at 50° C.without stirring.

[0185] 2nd Stage: Chlorine Stage (Stage C)

[0186] Each pulp suspension thereby obtained is then subjected to ableaching treatment which consists of a chlorination with chlorinewater. This treatment takes place on a pulp having a consistency of 3%as weight of dry matter.

[0187] To this end, an amount of chlorine of 2.89 (=0.17×17) %(weight/weight of dry pulp) is added to the enzyme-treated pulpsuspension and to one non-enzyme-treated pulp suspension. An amount ofchlorine of 3.40 (0.20×17) % (weight/weight) is added to the othernon-enzyme-treated pulp suspension.

[0188] The 3 suspensions are incubated for 1 hour at room temperature.The pulp is then washed with 40 volumes of demineralized water.

[0189] 3rd Stare: Sodium Hydroxide Stage (Stage E)

[0190] An alkaline extraction is then performed, which consists inadding 2% (weight/weight of dry pulp) of NaOH to the three suspensionsobtained above and which have a consistency of 5% as weight of drymatter.

[0191] The three suspensions are incubated for 1 hour 30 minutes at atemperature of 60° C. The pulp is then washed with 40 volumes ofdemineralized water and recovered in the form of a sheet having awhiteness of approximately 45° ISO (+/−3° ISO).

[0192] The Kappa number of the three sheets obtained is measured.

[0193] The Kappa number relates to the measurement of the amount oflignin present in the pulp. The Kappa number is a number whichrepresents the volume (in millilitres) of 0.1 N potassium permanganate(KMnO₄) solution consumed by one gram of dry pulp under the conditionsspecified and following the procedures described in TAPPI (TechnicalCommittee of the Association of the Pulp and Paper industry) standard#T236cm-85 (1985).

[0194] The degree ISO relates to the measurement of brightness of thepaper obtained from the pulp. This value is a factor of the reflectanceof the paper obtained from the pulp under the conditions specified andfollowing the procedures described in ISO (The InternationalOrganization for Standardization) standard #2469 published in standard#ISO 2470-1977 (F) supplementing standard #2470.

[0195] Four assays are carried out, which are identical except for theinitial pH adjustment to pH 5. In effect, four assays are carried outwith an aqueous suspension of pinewood pulp whose pH has been adjusted,respectively, to pH 6, pH 7, pH 8 and pH 9 instead of pH 5.

[0196] The results are collated in Table 3. TABLE 3 Amount of enzymes 50 0 employed (IU/g) Amount of chlorine 2.89 2.89 3.40 employed (% byweight/weight of dry pulp) Initial pH Kappa number 5 4.63 5.01 4.18 63.81 / 4.11 7 3.81 5.01 4.06 8 3.55 / 4.21 9 3.71 4.91 4.07

[0197] The symbol/means that the pulp suspension was not tested.

[0198] These results show that the xylanase according to the inventionpermits an approximately 15 to 20% reduction in the amount of chlorinefor a pulp bleached to 450 ISO. Furthermore, these results are obtainedboth at an alkaline pH and at an acid pH. These good results are alsoobtained at a pH of approximately 9.

[0199] This example shows that the xylanase according to the inventiondisplays activity over a wide pH range. In effect, the xylanaseaccording to the invention is active over a pH range betweenapproximately 5 and approximately 10. It is especially active for pHvalues above or equal to approximately 6. It is especially active for pHvalues below or equal to approximately 9.

EXAMPLE 12

[0200] Effect of Temperature on the Activity of the Xylanase Produced byBacillus sp. Strain 720/1 as an Aid to the Bleaching of Coniferous WoodPulp

[0201] Example 11 is repeated with 5 suspensions of coniferous wood pulpat pH 8. The enzyme treatment stage is carried out at a pH of 8 and atdifferent temperatures (55, 60 and 65° C.).

[0202] The results are collated in Table 4. TABLE 4 Amount of enzymes 50 0 employed (IU/g) Amount of chlorine 2.89 2.89 3.40 employed (% byweight/weight of dry pulp) Temperature ° C. Kappa number 55 3.89 / / 603.64 4.77 4.09 65 3.49 / /

[0203] The symbol/means that the pulp suspension was not tested.

[0204] It is observed that the pulp is bleached to approximately 45°ISO.

[0205] These results show that the Kappa number of the enzyme-treatedpulp samples remains well below the number of the non-enzyme-treatedsample.

[0206] This example shows that the xylanase according to the inventionis active over a wide temperature range. It is active at a temperatureof approximately 65° C.

[0207] This example also shows that the xylanase according to theinvention is stable at a temperature of approximately 60° C.

EXAMPLE 13

[0208] Activity of the Xylanase Produced by Bacillus sp. Strain 720/1 asan Aid to the Bleaching of Eucalyptus Pulp in the ECF Sequence

[0209] For this example, a eucalyptus pulp obtained from the companyCEASA Mill (Spain) is employed. The pulp is treated according to an ECF(“Elemental Chlorine Free”) sequence, that is to say the succession ofstages constituting the sequence does not make use of elementalchlorine.

[0210] 1st Stage: Oxygen Stage (Stage O)

[0211] The pulp is treated by a process employing oxygen as described inU.S. Pat. No. 4,462,864, such that a pulp having an initial Kappa numberof 12.3 and an initial degree ISO of 33.4 is obtained.

[0212] Two aqueous suspensions are prepared from this oxygen-treatedpulp having a consistency of 4% as weight of dry matter.

[0213] The pH of these suspensions is adjusted to pH 9 with HCl.

[0214] 2nd Stage: Enzyme Stage (Stage X)

[0215] The solution designated product N, as obtained in Example 2, isdiluted with demineralized water to obtain an enzyme solution having anenzyme activity of 25 IU/ml.

[0216] This enzyme solution containing the xylanase is added to onesuspension of pinewood pulp such that the pulp suspension is treated bymeans of 10 IU/g of dry pulp.

[0217] To the other two suspensions of pinewood pulp, demineralizedwater is added in place of the enzyme solution.

[0218] The 3 suspensions are then incubated for 1 hour 30 minutes at 50°C. without stirring.

[0219] 3rd Stage: Chlorine Dioxide Stage (Stage D)

[0220] Each pulp suspension thereby obtained is then subjected to ableaching treatment which consists of a chlorination with chlorinedioxide. This treatment takes place on a pulp having a consistency of 3%as weight of dry matter.

[0221] To this end, an amount of chlorine dioxide of 0.6% (weight/weightof dry pulp) is added to the enzyme-treated pulp suspension and to onenon-enzyme-treated pulp suspension. An amount of chlorine dioxide of 1%(weight/weight) is added to the other non-enzyme-treated pulpsuspension.

[0222] The 3 suspensions are incubated for 30 minutes at 50° C. The pulpis then washed with 40 volumes of demineralized water.

[0223] 4th Stage: Sodium Hydroxide/Hydrogen Peroxide Stage (Stage E/P)

[0224] An alkaline extraction is then performed, which consists inadding 1.8% (weight/weight of dry pulp) of NaOH and 0.5% (weight/weightof dry pulp) of hydrogen peroxide to the three suspensions obtainedabove, and which have a consistency of 12% as weight of dry matter.

[0225] The three suspensions are incubated for 1 hour 30 minutes at atemperature of 70° C. The pulp is then washed with 40 volumes ofdemineralized water.

[0226] 5th Stage: Chlorine Dioxide Stage (Stage D)

[0227] Each pulp suspension thereby obtained is then subjected again toa bleaching treatment which consists of a chlorination with chlorinedioxide. This treatment takes place on a pulp having a consistency of12%.

[0228] An amount of chlorine dioxide of 0.5% (weight/weight of dry pulp)is added to these three suspensions.

[0229] The 3 suspensions are incubated for 2 hours at 75° C. The pulp isthen washed with 40 volumes of demineralized water.

[0230] 6th Stage: Sodium Hydroxide/Hydrogen Peroxide Stage (Stage E/P)

[0231] An alkaline extraction is then performed, which consists inadding 0.6% (weight/weight of dry pulp) of NaOH and 0.3% (weight/weightof dry pulp) hydrogen peroxide to the three suspensions obtained above,and which have a consistency of 12% as weight of dry matter.

[0232] The three suspensions are incubated for 1 hour 30 minutes at atemperature of 70° C. The pulp is then washed with 40 volumes ofdemineralized water.

[0233] 7th Stage: Chlorine Dioxide Stage (Stage D)

[0234] Each pulp suspension thereby obtained is then subjected to ableaching treatment which consists of a chlorination with chlorinedioxide. This treatment takes place on a pulp having a consistency of12%.

[0235] An amount of chlorine dioxide of 0.3% (weight/weight) is added tothese three suspensions.

[0236] The 3 suspensions are incubated for 2 hours 30 minutes at 75° C.The pulp is then washed with 40 volumes of demineralized water and isrecovered in the form of a sheet.

[0237] The degree ISO of the three sheets obtained is measured.

[0238] The results are collated in Table 5. TABLE 5 Amount of enzymes 100 0 employed in stage 2 in IU/g Amount of chlorine dioxide employed in0.6 0.6 1.0 stage 3 in % (weight/weight of dry pulp) °ISO 88.5 85.5 87.9

[0239] This example shows that the xylanase according to the inventionis effective on eucalyptus pulp. Furthermore, it does not necessitateany pH adjustment, since it has the advantage of being active at the pHof the pulp, that is to say at a pH of approximately 9. This exampleshows that the xylanase according to the invention is an alkalinexylanase.

[0240] This example also shows that, in comparison with what is obtainedwithout xylanase, the use of the xylanase according to the inventionbrings about an increase in brightness of at least 3° ISO for a fixedamount of ClO₂.

[0241] This example also shows that the use of the xylanase according tothe invention enables the amount of ClO₂ to be reduced by approximately4 to 5 kg/tonne of pulp, representing approximately 25 to 30% of thetotal amount of ClO₂ needed.

EXAMPLE 14

[0242] Determination of the Nucleotide and Protein Sequence of Bacillussp. 720/1 Xylanase

[0243] 1. Extraction of Chromosomal DNA from Bacillus sp. Strain 720/1

[0244] From the culture B as obtained in Example 2, culturing of 200 mlof Bacillus sp. strain 720/1 is carried out in LB/C medium for 16 hoursat 37° C. LB/C medium is identical to the LBS/C medium described inExample 1, without the addition of wheat bran.

[0245] When this culture has been prepared and is in stationary phase,it is centrifuged (Beckman J 21, JA10 rotor) at 5,000 rpm for 10minutes. The centrifugation pellet thereby obtained is taken up in 9 mlof Tris-HCl (tris(hydroxymethyl)aminomethane acidified with 0.1 M HCl)buffer at pH 8, 0.1 M EDTA (ethylenediaminetetra-acetic acid), 0.15 MNaCl containing 18 mg of lysozyme; the suspension thereby obtained isincubated for 15 minutes at 37° C.

[0246] The lysate thereby obtained is then treated with 200 μl of anRNAse solution at a concentration of 10 mg/ml for 20 minutes at 50° C. 1ml of 10% (w/v) SDS (sodium dodecyl sulphate) solution is then added tothis lysate. This lysate is then incubated for 30 minutes at 70° C.

[0247] The lysate is thereafter cooled to around 45° C., and 0.5 ml of asolution of proteinase K (sold by Boehringer Mannheim) at aconcentration of 20 mg/ml (prepared immediately before use) is thenadded to it.

[0248] The lysate is incubated at 45° C. with stirring until atransparent solution is obtained.

[0249] Several phenol extractions are performed on this transparentsolution under the conditions and following the procedures described inMolecular Cloning—a laboratory manual—Sambrook, Fritsch, Maniatis—secondedition, 1989, on page E.3, until a clean interface is obtained, asdescribed therein.

[0250] The DNA is precipitated with 20 ml of ethanol. The precipitate isrecovered by centrifugation at 5,000 rpm (Beckman J21, JA10 rotor) for 5minutes, and then suspended in 2 ml of TE buffer, pH 8.0, (10 mMTris-HCl, 1 mM EDTA, pH 8.0). This suspension contains the chromosomalDNA.

[0251] 2. Construction of the Vector pUBR2002

[0252] The vector pUBR2002 (E. coli-Bacillus subtilis) was obtained fromplasmid pBR322 which is sold by the company Biolabs (ClontechLaboratories catalogue No. 6210-1) and the vector pUB131.

[0253] Two synthetic oligonucleotides are constructed by the techniquedescribed in Beaucage et al. (1981), Tetrahedron Letters, 22, pages1859-1882 and using β-cyanoethyl phosphoramidites in a Biosearch CycloneSynthesizer.

[0254] The sequences of these two oligonucleotides are as follows:

[0255] SEQ ID NO:14

[0256] 5′-CCCCCCTACGTAGCGGCCGCCCCGGCCGGTAACCTAGGAAGTCAGCGCCCTGCACC-3′and SEQ ID NO:15

[0257] 5′-CCCCCCTACGTAGGCCGGGGCGGCCGCGGTTACCTAGGGCCTCGTGATACGCCTAT-3′

[0258] These two oligonucleotides are used to perform a PCRamplification on plasmid pBR322 according to the technique described inMolecular Cloning, a laboratory Manual—Sambrook et al., second edition,1989, pages 14.18-14.19.

[0259] The PCR-amplified fragment contains the E. coli replicon limitedon both sides by the AvrII, BstEII, NotI, SfiI, SnabI restriction sites.

[0260] The approximately 2.8-kbp (kbp=1,000 base pairs) SnabI-SnabIfragment is ligated with the vector pUB131 which has previously beensubjected to digestion with SnabI. Construction of the vector pUB131 isdescribed in Example 10 and in FIG. 7 of U.S. Pat. No. 5,352,603(European Patent Application 0,415,296), which is incorporated byreference.

[0261] The ligation technique is described by Sambrook et al. (pages1.68-1.69). All the ligations carried out in the examples in thisapplication were performed according to this technique.

[0262] The ligation thereby obtained is transformed into E. coli MC1061cells [Clontech Laboratories, catalogue No. C-1070-1] by electroporation(Sambrook et al., pages 1.75-1.81). The transformed cells are culturedon Petri dishes containing LB agar medium, 100 μg/ml of ampicillin and10 μg/ml of kanamycin, at 37° C. for approximately 18 hours.

[0263] The plasmids are extracted from the colonies isolated by thealkaline lysis method (Sambrook et al., pages 1.25-1.28) and aresubjected to a restriction analysis, the analysis described in MolecularCloning, a laboratory Manual—Maniatis et al., 1982, Cold Spring HarborLaboratory, pages 374-379.

[0264] A strain is obtained from which the vector which is designatedpUBR2002 (FIG. 3) is extracted.

[0265] 3. Construction of a Bacillus sp. 720/1 Gene Library

[0266] From the suspension containing it, the chromosomal DNA ispartially cleaved with the restriction enzyme Sau3AI. The restrictionconditions are those described by Sambrook et al. (pages 5.28-5.32),except that these restriction conditions are increased by a factor of 10in order to obtain a sufficient amount of DNA for the followingpurification steps.

[0267] The ratio of the amount of DNA employed to the amount of enzymeis adjusted in order to obtain a maximum of fragments between 4 and 7kbp (kbp: 10³ base pairs) in size.

[0268] The set of fragments thereby obtained is then subjected toagarose (0.8% w/v) gel electrophoresis as described by Sambrook et al.,pages 6.01-6.19, and the fragments between 4 and 7 kbp in size areisolated and purified by the method of filtration followed bycentrifugation described in Zhu et al., Bio/Technology 3, 1985, pages1014-1016.

[0269] The DNA fragments thus purified are then ligated (according tothe method described by Sambrook et al., (pages 1.68-1.69) with plasmidpUBR2002 (E. coli-Bacillus subtilis) previously cut at the BamHI siteand dephosphorylated as described by Sambrook et al., (pages 1.60-1.61).

[0270] The ligation thereby obtained is used to transform E. coli MC1061cells by electroporation (Sambrook et al., pages 1.75-1.81).

[0271] 4. Screening of the Gene Library

[0272] The transformed E. coli cells are cultured on Petri dishescontaining LB agar medium, 0.8 g/l of AZCL-xylan and 100 μg/ml ofampicillin, for approximately 24 hours at 37° C. A colony displaying azone of hydrolysis is isolated.

[0273] The plasmid present in this colony is extracted and isolated bythe alkaline lysis technique described in Sambrook et al., pages1.25-1.28.

[0274] A restriction analysis (Sambrook et al., page 1.85) is performed.This analysis shows that the DNA fragment obtained, which contains thexylanase gene, is approximately 3.5 kbp (kbp=1,000 base pairs) in size.It is carried by the vector pUBR2002 which has been ligated.

[0275] The plasmid pUBR-720X1 (FIG. 4) is thereby obtained.

[0276] 5. Cloning of a Chromosomal Fragment Containing the Xylanase Gene

[0277] Plasmid pUBR-720X1 is digested with restriction enzymes at theSwaI and SpeI sites. The xylanase gene is thereby obtained on anapproximately 1.5-kbp Swal-SpeI DNA fragment.

[0278] This SwaI-SpeI DNA fragment is subjected to a treatment with theKlenow fragment of DNA polymerase (Sambrook et al., pages F.2-F.3).

[0279] The DNA preparation thereby obtained is ligated with the vectorpUBR2002 which has previously been digested with-EcoRV anddephosphorylated.

[0280] The ligation is then transformed into E. coli MC1061 cells byelectroporation.

[0281] The transformed strains are selected on Petri dishes containingLB (Luria-Bertani) agar medium, 0.8 g/l of AZCL-xylan (Megazyme) and 100μg/ml of ampicillin, after growth at 37° C. for approximately 24 hours.

[0282] Colonies displaying a zone of hydrolysis are isolated. Theplasmids are extracted from the colonies isolated by the alkaline lysistechnique (Sambrook et al., pages 1.25-1.28), and are subjected to arestriction analysis (Maniatis et al., 1982, pages 374-379). Thisrestriction analysis shows that the plasmid isolated, pUBR-720X11 (FIG.5), contains the xylanase gene on an approximately 1.5 kbp fragment ofthe Bacillus sp. 720/1 chromosomal DNA.

[0283] Plasmid pUBR-720X11 is then used to transform E. coli JM109 cells(Clontech Laboratories catalogue No. C1005-1) by the CaCl₂ technique(Sambrook et al., pages 1.82-1.84).

[0284] The transformed E. coli cells are cultured on Petri dishescontaining LB agar medium, 0.8 g/l AZCL-xylan and 100 μg/ml ofampicillin. After growth at 37° C. for approximately 24 hours, a zone ofhydrolysis is observed around the colonies. This shows that thetransformed E. coli cells do indeed express the xylanase.

[0285] The technique enabling the DNA fragments to be dephosphorylatedor the vectors to be linearized is described by Sambrook et al., (pages1.60-1.61).

[0286] A colony displaying a zone of hydrolysis is isolated. The plasmidpresent in this colony is extracted and isolated by the alkaline lysistechnique.

[0287] The sequence of the xylanase is established using the methoddescribed in Sambrook et al. pages 13.15 and 13.17 (FIG. 13.3B), usingplasmid pUBR-720X11 as template.

[0288] To initiate the sequence determination, syntheticoligonucleotides are prepared for hybridization with plasmid pUBR2002.The sequences of these synthetic ologonucleotides are as follows:

[0289] SEQ ID NO:16

[0290] 5′-ACGAGGAAAGATGCTGTTCTTGTAAATGAGT-3′

[0291] and

[0292] SEQ ID NO:17

[0293] 5′-TACCTTGTCTACAAACCCC-3′

[0294] The remainder of the sequence is determined by the use of othersynthetic oligonucleotides chosen on the basis of the newly determinedportions of the sequence.

[0295] The nucleotide sequence of the complete gene which codes forxylanase (SEQ ID NO:10) is thereby obtained (FIG. 2). The xylanase geneis obtained as an approximately 1.5-kbp fragment.

EXAMPLE 15

[0296] Construction of the Expression Vector pUBRD-720X11

[0297] The expression vector pUBRD-720X11 (FIG. 6) is a plasmid derivedfrom plasmid pUBR-720X11 from which the E. coli replicon has beenremoved.

[0298] The construction of plasmid pUBRD-720X11 from plasmidpUBR-720X11, as obtained in Example 14, is described below.

[0299] Plasmid pUBR-720X11 is digested with the restriction enzyme atthe SnaBI sites for the purpose of removing the origin of replication ofE. coli, according to the technique described in Example 14. Anapproximately 5-kbp fragment is thereby obtained; it is ligated withitself according to the technique described in Example 14, to obtainplasmid pUBRD-720X11.

[0300] The ligation thereby obtained is used to transform competentBacillus subtilis SE3 cells according to the technique described in DNACloning, vol. II, ed. Glover, D. M., IRL Press Oxford, 1985, pages 9-11.

[0301]Bacillus subtilis strain SE3 was deposited on Jun. 21,st 1993 atthe collection named Belgian Coordinated Collections of Microorganisms(LMG culture collection, Ghent, Belgium) in accordance with the BudapestTreaty under the number LMG P-14035.

[0302] The transformed cells are cultured on Petri dishes containing LB(Luria-Bertani) agar medium, 0.8 g/l of AZCL-xylan and 25 μg/ml ofkanamycin, at 37° C. for approximately 18 hours. After growth, coloniesdisplaying a zone of hydrolysis are isolated.

[0303] The isolated colonies are subjected to a plasmid analysis byenzyme restriction for the purpose of verifying that the construction ofthe plasmid is correct, according to the technique described in Example14.

[0304] A strain is obtained from which the vector which is designatedpUBRD-720X11 may be isolated.

EXAMPLE 16

[0305] Transformation of Bacillus licheniformis Strain SE2 delap6 withthe Expression Vector pUBRD-720X11

[0306] Plasmid pUBRD-720X11 described in Example 15 is extracted fromits host, isolated and purified (Sambrook et al., 1989, p. 1.25-1.28).

[0307] A culture of Bacillus licheniformis strain SE2 delap6 isprepared. Bacillus licheniformis strain SE2 delap6 and the culturingthereof are described in Examples 27 and 28 of European PatentApplication 0,634,490, which is incorporated by reference.

[0308]Bacillus licheniformis strain SE2 delap6 was prepared fromBacillus licheniformis strain SE2, which was deposited on Jun. 21, 1993at the collection named Belgian Coordinated Collections ofMicroorganisms (LMG culture collection, Ghent, Belgium) in accordancewith the Budapest Treaty under the number LMG P-14034.

[0309]Bacillus licheniformis strain SE2 delap6 is then transformed withplasmid pUBRD-720X11 according to the protoplast technique (Maniatis etal., p. 150-151).

[0310] The transformed strain [Bacillus licheniformis SE2 delap6(pUBRD-720X11)] is selected on Petri dishes containing LB agar medium,0.8 g/l of AZCL-xylan and 25 μg/ml of kanamycin. It is then isolated andpurified by screening, that is to say by being applied and streaked toobtain single colonies at the surface of LB (Luria-Bertani) agar mediumwhich is described in Molecular Cloning—Laboratory Manual (Sambrook etal.), 1989, p. A.4.

EXAMPLE 17

[0311] Production of Xylanase by B. licheniformis SE2 delap6(pUBRD-720X11)

[0312]B. licheniformis strain SE2 delap6 transformed by plasmidpUBRD-720X11, as obtained in Example 16, is cultured for 17 hours at 37°C. in an LB culture medium supplemented with 0.5% (w/v) of glucose and20 μg/ml of kanamycin.

[0313] This culture is transferred (5% v/v) to 50 ml of M2 mediumsupplemented with 20 μg/ml of kanamycin.

[0314] M2 medium contains 30 g of soya flour, 75 g of soluble starch, 2g of sodium sulphate, 5 mg of magnesium chloride, 3 g of NaH₂PO₄, 0.2 gof CaCl₂.H₂O and 1,000 ml of water. The pH of this M2 medium is adjustedto 5.8 with 10 N NaOH before it is sterilized.

[0315] The culture is incubated with orbital shaking at the rate of 250rpm with an amplitude of approximately 2.54 cm for 80 hours at 37° C.After 80 hours, the biomass is removed by centrifugation (Beckman J21,JA10 rotor) at 5,000 rpm for 10 minutes. The centrifugation supernatantis retained. The enzyme activity is measured on this supernatantaccording to the technique described in Example 2, and the presence ofan xylanase activity is noted.

EXAMPLE 18

[0316] Construction of the Vector pUBC2001

[0317] The vector pUBC2001 (E. coli-Bacillus) (FIG. 7) is a plasmidderived from the plasmid pUBC131 containing, as sole difference, thepresence of two additional restriction sites: BstEII and PacI.Construction of the vector UBC131 is described in Example 11 and in FIG.8 of U.S. Pat. No. 5,352,603 (European Patent Application 0,415,296),which is incorporated by reference.

[0318] The construction of this plasmid is described below.

[0319] Four synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

[0320] The sequences of these four oligonucleotides are as follows:

[0321] SEQ ID NO: 18

[0322] 5′-CGGTCGCCGCATACACTA-3′

[0323] SEQ ID NO: 19

[0324] 5′-CCCCCCCCCGGTAACCTGCATTAATGAATCGGCCAA-3′

[0325] SEQ ID NO: 20

[0326] 5′-CCCCCCCCCGGTTACCGTATTTATTAACTTCTCCTAGTA-3′

[0327] SEQ ID NO: 21

[0328] 5′-CCCCCCTCTAGATTAATTAACCAAGCTTGGGATCCGTCGACCTGCAGATC-3′

[0329] The two oligonucleotides having the sequences SEQ ID NO: 18 and19 are used to perform a PCR amplification on the vector pUBC131according to the PCR technique described in-Example 14. ThePCR-amplified fragment, containing a portion of the ampicillin resistantgene and the functions needed for replication in E. coli, is subjectedto a restriction with ScaI and BstEII, generating an approximately1.5-1.6-kbp fragment.

[0330] A second PCR amplification is carried out on the vector pUBC131,using the oligonucleotides having the sequences SEQ ID NO: 20 and 21 andaccording to the technique described in Example 14. The PCR-amplifiedfragment contains a portion of the vector pUBC131. This fragment issubjected to a restriction with BstEII and EcoRI, generating anapproximately 1.4-1.5-kbp fragment.

[0331] The two fragments thereby obtained are ligated together,according to the technique described in Example 14, with the vectorpUBC131 which has previously been subjected to a double digestion withEcoRI and ScaI, according to the technique described in Example 14.

[0332] The ligation thereby obtained is used to transform E. coli MC1061cells by electroporation according to the technique described in Example14. The transformed cells are cultured on Petri dishes containing LBagar medium, 100 μg/ml of ampicillin and 10 μg/ml of kanamycin, at 37°C. for approximately 18 hours.

[0333] After growth, the isolated colonies are subjected to a plasmidanalysis by enzyme restriction according to the technique described inExample 14.

[0334] A strain is obtained from which the vector which is designatedpUBC2001 may be extracted.

EXAMPLE 19

[0335] Construction of the Expression Vector pC-BPX-PRE-2003

[0336] The expression vector pC-BPX-PRE-2003 (E. coli-Bacillus) (FIG. 8)is an expression vector derived from plasmid pUBC2001. It contains thepromoter derived from the gene which codes for Bacillus pumilus PRL B12xylanase and the presequence which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase. The method for preparing andobtaining the promoter derived from the gene which codes for Bacilluspumilus PRL B12 xylanase and the presequence which codes for the signalpeptide of Bacillus pumilus PRL B12 xylanase is described in Example 17and in FIG. 1 of European Patent Application 0,634,490, which isincorporated in this application by reference.

[0337] The sequence of the promoter derived from the gene which codesfor Bacillus pumilus PRL B12 xylanase is described in the presentapplication under the number SEQ ID NO: 26. The sequence of thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase is described in the present application under the numberSEQ ID NO: 27.

[0338] The construction of plasmid pC-BPX-PRE-2003 is described below.

[0339] Two synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

[0340] The sequences of these two oligonucleotides are as follows:

[0341] SEQ ID NO: 22

[0342] 5′-CCCCCCTGAAATCAGCTGGACTAAAAGGGATGCAATTTC-3′

[0343] SEQ ID NO: 23

[0344] 5′-CCCCCCGTCGACCGCATGCGCCGGCACAGC-3′

[0345] These two oligonucleotides are used to perform a PCRamplification on the plasmid pUB-BPX12 according to the techniquedescribed in Example 14. Construction of the plasmid pUB-BPX12 isdescribed in Example 17 and in FIG. 4 of European Patent Application0,634,490, which is incorporated by reference.

[0346] The use of the oligonucleotide having the sequence SEQ ID NO: 22makes it possible, by changing one nucleotide, to remove, upstream ofthe B. pumilus PRL B12 xylanase promoter, the SphI restriction site,located at approximately 5.5 kbp, normally present in pUBC2001. Thechange of nucleotide is represented by the nucleotide underlined in thesequence SEQ ID NO: 22 above, and relates to the replacement of C by Gfor the SphI site (SphI=GCATGC).

[0347] The sequence SEQ ID NO: 23 enables a new SphI site to be createdat the end of the presequence which codes for the signal peptide of B.pumilus PRL B12 xylanase, by changing only one nucleotide of the Alacodon [25], that is to say by changing GCG to GCT.

[0348] The PCR amplified fragment, containing the promoter derived fromthe gene which codes for B. pumilus PRL B12 xylanase and the presequencewhich codes for the signal peptide of B. pumilus PRL B12 xylanase, issubjected to a restriction with PvuII and SphI, generating anapproximately 0.7-kbp fragment, according to the technique described inExample 14.

[0349] The approximately 0.7-kbp PvuII-SphI fragment is ligated with thevector pUBC2001 which has previously been subjected to a doubledigestion with PvuII and SphI, according to the techniques described inExample 14.

[0350] The ligation thereby obtained is used to transform E. coli MC1061cells by electroporation according to the technique described in Example14. The transformed cells are cultured on Petri dishes containing LBagar medium, 100 μg/ml of ampicillin, at 37° C. for approximately 18hours.

[0351] After growth, the isolated colonies are subjected to a plasmidanalysis by enzyme restriction according to the technique described inExample 14.

[0352] A strain is obtained from which the vector which is designatedpC-BPX-PRE-2003 may be extracted.

EXAMPLE 20

[0353] Construction of the Expression Vector PC-BPX-PRE-720X

[0354] The expression vector pC-BPX-PRE-720X (E. coli-Bacillus) (FIG. 9)is an expression vector containing the promoter derived from the genewhich codes for B. pumilus PRL B12 xylanase and the presequence whichcodes for the signal peptide of B. pumilus PRL B12 xylanase and thesequence of the gene which codes for the mature portion of Bacillus sp.720/1 xylanase.

[0355] The expression vector pC-BPX-PRE-720X contains the sequence SEQID NO: 1, the nucleotide sequence of the gene which codes for the matureportion of Bacillus sp. 720/1 xylanase.

[0356] The construction of plasmid pC-BPX-PRE-720X is described below.

[0357] Two synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

[0358] The sequences of these two oligonucleotides are as follows:

[0359] SEQ ID NO: 24

[0360] 5′-CCCCCCGCATGCGCAAATCGTCACCGACAATTCCATTGG-3′

[0361] SEQ ID NO: 25

[0362] 5′-TACCTTGTCTACAAACCCC-3′

[0363] These two oligonucleotides are used to perform a PCRamplification on plasmid pUBR-720X11, as obtained in Example 14, andaccording to the technique described in Example 14.

[0364] The PCR amplified fragment containing the sequence of gene whichcodes for the mature portion of Bacillus sp. 720/1 xylanase is subjectedto a restriction with SphI and SacI, generating an approximately 0.8-kbpfragment, according to the technique described in Example 14.

[0365] The SphI-SacI fragment is ligated with the vector pC-BPX-PRE-2003which has previously been subjected to a double digestion with SphI andSacI, according to techniques described in Example 14. Ligation at theSphI restriction site enables a translational fusion of the signalsequence of the gene which codes for B. pumilus PRL B12 xylanase withthe sequence of the gene which codes for the mature portion of Bacillussp. 720/1 xylanase to be created.

[0366] The ligation thereby obtained is used to transform E. coli MC1061cells by electroporation according to the technique described in Example14. The transformed cells are cultured on Petri dishes containing LBagar medium, 0.8 g/l of AZCL-xylan and 100 μg/ml of ampicillin, at 37°C. for approximately 18 hours. Colonies displaying a zone of hydrolysisare isolated.

[0367] After growth, the isolated colonies are subjected to a plasmidanalysis by enzyme restriction according to the technique described inExample 14.

[0368] A strain is obtained from which the vector which isdesignated-pC-BPX-PRE-720X may be extracted.

EXAMPLE 21

[0369] Construction of the Vector PBPXD-PRE-720X

[0370] The vector pBPXD-PRE-720X (Bacillus) (FIG. 10) is an expressionvector derived from plasmid pUB131. It contains the promoter derivedfrom the gene which codes for B. pumilus PRL B12 xylanase and thepresequence which codes for the signal peptide of B. pumilus PRL B12xylanase and the sequence of the gene which codes for the mature portionof Bacillus sp. 720/1 xylanase.

[0371] The construction of plasmid pBPXD-PRE-720X is described below.

[0372] Plasmid pC-BPX-PRE-720X obtained in Example 20 is subjected to arestriction with PvuII and EcoRI, generating an approximately 1.5-kbpfragment, according to the technique described in Example 14.

[0373] The approximately 1.5-kbp fragment is ligated with the vectorpUB131 which has previously been subjected to a double digestion withPvuII and EcoRI, according to techniques described in Example 14.

[0374] The ligation thereby obtained is used to transform form B.subtilis SE3 cells according to the electroporation technique describedin Example 14. The transformed cells are cultured on Petri dishescontaining LB agar medium, 0.8 g/l of AZCL-xylan and 25 μg/ml ofkanamycin, at 37° C. for approximately 18 hours. Colonies displaying abroad zone of hydrolysis are isolated.

[0375] After growth, the isolated colonies are subjected to a plasmidanalysis by enzyme restriction according to the technique described inExample 14.

[0376] A strain is thereby obtained from which the vector which isdesignated pBPXD-PRE-720X may be extracted.

EXAMPLE 22

[0377] Transformation of Bacillus licheniformis SE2 delap6 with theExpression Vector pBPXD-PRE-720X

[0378] Plasmid pBPXD-PRE-720X (FIG. 10) described in Example 21 isextracted from its host, isolated and purified.

[0379] A culture of B. licheniformis strain SE2 delap6 is preparedaccording to the technique described in Example 16. This strain is thentransformed with this plasmid according to the protoplast techniquedescribed in Example 16.

[0380] The transformed strain [B. licheniformis SE2 delap6(pBPXD-PRE-720X)] is selected from Petri dishes containing LB agarmedium, 0.8 g/l of AZCL-xylan and 25 μ/ml of kanamycin. The strain isisolated and purified by screening according to the technique describedin Example 16.

EXAMPLE 23

[0381] Production of Xylanase by B. licheniformis SE2 delap6(pBPXD-PRE-720X)

[0382] An assay is performed which is identical to the one described inExample 17, but with B. licheniformis strain SE2 delap6 transformed byplasmid pBPXD-PRE-720X as obtained in Example 22.

[0383] The enzyme activity is measured according to the techniquedescribed in Example 2 on the supernatant obtained, and the presence ofa xylanase activity is noted.

EXAMPLE 24

[0384] Preparation and Isolation of the Xylanase Produced by B.licheniformis Strain SE2 delap6 (pBPXD-PRE-720X)

[0385] The xylanase produced by B. licheniformis strain SE2 delap6transformed by plasmid pBPXD-PRE-720X, as obtained in Example 22, isisolated and purified. This strain is cultured according to the protocoldescribed in Example 23.

[0386] The xylanase obtained is isolated and purified according to theprotocol described in Example 3 of European Patent Application0,634,490, which is incorporated in this application by reference.

EXAMPLE 25

[0387] Amino Acid Sequence of the Xylanase Produced by B. licheniformisStrain SE2 delap6 (pBPXD-PRE-720X)

[0388] The sequence of the first 50 amino acids of the xylanase producedby B. licheniformis strain SE2 delap6 (pBPXD-PRE-720X) is determinedusing a sequencing apparatus (HP G1000A, Hewlett-Packard) and accordingto the instruction leaflet of this apparatus.

[0389] The xylanase isolated and purified as described in Example 24 isused.

[0390] It is verified that the sequence obtained is identical to the onedescribed in Example 4 for the xylanase produced by Bacillus sp. strain720/1.

EXAMPLE 26

[0391] Determination of the Molecular Weight of the Xylanase Produced byB. licheniformis Strain SE2 delap6 (pBPXD-PRE-720X)

[0392] The molecular weight of the xylanase produced by B. licheniformisstrain SE2 delap6 (pBPXD-PRE-720X) is determined according to theprotocol described in Example 7 and employing the xylanase isolated andpurified as described in Example 24.

[0393] Staining with Coomassie blue reveals a polypeptide of molecularweight between 25 and 26 kD, which is identical to that of the xylanaseproduced by Bacillus sp. strain 720/1.

1 29 663 base pairs nucleic acid single linear DNA (genomic) Bacillus 1CAAATCGTCA CCGACAATTC CATTGGCAAC CACGATGGCT ATGATTATGA ATTTTGGAAA 60GATAGCGGTG GCTCTGGGAC AATGATTCTC AATCATGGCG GTACGTTCAG TGCCCAATGG 120AACAATGTTA ACAACATATT ATTCCGTAAA GGTAAAAAAT TCAATGAAAC ACAAACACAC 180CAACAAGTTG GTAACATGTC CATAAACTAC GGAGCCAACT TCCAACCAAA TGGTAATGCG 240TATTTATGCG TCTATGGTTG GACTGTTGAC CCTCTTGTCG AATATTATAT TGTCGACAGT 300TGGGGCAACT GGCGTCCACC AGGAGCAACG CCTAAGGGGA CCATCACTGT TGATGGAGGA 360ACATATGATA TCTACGAGAC TCTTAGAGTC AATCAACCCT CCATTAAGGG GATTGCCACA 420TTTAAACAAT ATTGGAGTGT TCGAAGATCG AAACGCACGA GTGGCACGAT TTCTGTCAGC 480AACCACTTTA GAGCGTGGGA AAACTTAGGG ATGAATATGG GGAAAATGTA TGAAGTCGCG 540CTTACTGTAG AAGGCTATCA AAGTAGCGGA AGTGCTAATG TATATAGCAA TACACTAAGA 600ATTAACGGTA ACCCTCTCTC AACTATTAGT AATGACGAGA GCATAACTTT GGATAAAAAC 660AAT 663 663 base pairs nucleic acid single linear DNA (genomic) Bacillusmat_peptide 1..663 CDS 1..663 2 CAA ATC GTC ACC GAC AAT TCC ATT GGC AACCAC GAT GGC TAT GAT TAT 48 Gln Ile Val Thr Asp Asn Ser Ile Gly Asn HisAsp Gly Tyr Asp Tyr 1 5 10 15 GAA TTT TGG AAA GAT AGC GGT GGC TCT GGGACA ATG ATT CTC AAT CAT 96 Glu Phe Trp Lys Asp Ser Gly Gly Ser Gly ThrMet Ile Leu Asn His 20 25 30 GGC GGT ACG TTC AGT GCC CAA TGG AAC AAT GTTAAC AAC ATA TTA TTC 144 Gly Gly Thr Phe Ser Ala Gln Trp Asn Asn Val AsnAsn Ile Leu Phe 35 40 45 CGT AAA GGT AAA AAA TTC AAT GAA ACA CAA ACA CACCAA CAA GTT GGT 192 Arg Lys Gly Lys Lys Phe Asn Glu Thr Gln Thr His GlnGln Val Gly 50 55 60 AAC ATG TCC ATA AAC TAC GGA GCC AAC TTC CAA CCA AATGGT AAT GCG 240 Asn Met Ser Ile Asn Tyr Gly Ala Asn Phe Gln Pro Asn GlyAsn Ala 65 70 75 80 TAT TTA TGC GTC TAT GGT TGG ACT GTT GAC CCT CTT GTCGAA TAT TAT 288 Tyr Leu Cys Val Tyr Gly Trp Thr Val Asp Pro Leu Val GluTyr Tyr 85 90 95 ATT GTC GAC AGT TGG GGC AAC TGG CGT CCA CCA GGA GCA ACGCCT AAG 336 Ile Val Asp Ser Trp Gly Asn Trp Arg Pro Pro Gly Ala Thr ProLys 100 105 110 GGG ACC ATC ACT GTT GAT GGA GGA ACA TAT GAT ATC TAC GAGACT CTT 384 Gly Thr Ile Thr Val Asp Gly Gly Thr Tyr Asp Ile Tyr Glu ThrLeu 115 120 125 AGA GTC AAT CAA CCC TCC ATT AAG GGG ATT GCC ACA TTT AAACAA TAT 432 Arg Val Asn Gln Pro Ser Ile Lys Gly Ile Ala Thr Phe Lys GlnTyr 130 135 140 TGG AGT GTT CGA AGA TCG AAA CGC ACG AGT GGC ACG ATT TCTGTC AGC 480 Trp Ser Val Arg Arg Ser Lys Arg Thr Ser Gly Thr Ile Ser ValSer 145 150 155 160 AAC CAC TTT AGA GCG TGG GAA AAC TTA GGG ATG AAT ATGGGG AAA ATG 528 Asn His Phe Arg Ala Trp Glu Asn Leu Gly Met Asn Met GlyLys Met 165 170 175 TAT GAA GTC GCG CTT ACT GTA GAA GGC TAT CAA AGT AGCGGA AGT GCT 576 Tyr Glu Val Ala Leu Thr Val Glu Gly Tyr Gln Ser Ser GlySer Ala 180 185 190 AAT GTA TAT AGC AAT ACA CTA AGA ATT AAC GGT AAC CCTCTC TCA ACT 624 Asn Val Tyr Ser Asn Thr Leu Arg Ile Asn Gly Asn Pro LeuSer Thr 195 200 205 ATT AGT AAT GAC GAG AGC ATA ACT TTG GAT AAA AAC AAT663 Ile Ser Asn Asp Glu Ser Ile Thr Leu Asp Lys Asn Asn 210 215 220 221amino acids amino acid linear protein 3 Gln Ile Val Thr Asp Asn Ser IleGly Asn His Asp Gly Tyr Asp Tyr 1 5 10 15 Glu Phe Trp Lys Asp Ser GlyGly Ser Gly Thr Met Ile Leu Asn His 20 25 30 Gly Gly Thr Phe Ser Ala GlnTrp Asn Asn Val Asn Asn Ile Leu Phe 35 40 45 Arg Lys Gly Lys Lys Phe AsnGlu Thr Gln Thr His Gln Gln Val Gly 50 55 60 Asn Met Ser Ile Asn Tyr GlyAla Asn Phe Gln Pro Asn Gly Asn Ala 65 70 75 80 Tyr Leu Cys Val Tyr GlyTrp Thr Val Asp Pro Leu Val Glu Tyr Tyr 85 90 95 Ile Val Asp Ser Trp GlyAsn Trp Arg Pro Pro Gly Ala Thr Pro Lys 100 105 110 Gly Thr Ile Thr ValAsp Gly Gly Thr Tyr Asp Ile Tyr Glu Thr Leu 115 120 125 Arg Val Asn GlnPro Ser Ile Lys Gly Ile Ala Thr Phe Lys Gln Tyr 130 135 140 Trp Ser ValArg Arg Ser Lys Arg Thr Ser Gly Thr Ile Ser Val Ser 145 150 155 160 AsnHis Phe Arg Ala Trp Glu Asn Leu Gly Met Asn Met Gly Lys Met 165 170 175Tyr Glu Val Ala Leu Thr Val Glu Gly Tyr Gln Ser Ser Gly Ser Ala 180 185190 Asn Val Tyr Ser Asn Thr Leu Arg Ile Asn Gly Asn Pro Leu Ser Thr 195200 205 Ile Ser Asn Asp Glu Ser Ile Thr Leu Asp Lys Asn Asn 210 215 220744 base pairs nucleic acid single linear DNA (genomic) Bacillus 4ATGAGACAAA AGAAATTGAC GTTGATTTTA GCCTTTTTAG TTTGTTTTGC ACTAACCTTA 60CCTGCAGAAA TAATTCAGGC ACAAATCGTC ACCGACAATT CCATTGGCAA CCACGATGGC 120TATGATTATG AATTTTGGAA AGATAGCGGT GGCTCTGGGA CAATGATTCT CAATCATGGC 180GGTACGTTCA GTGCCCAATG GAACAATGTT AACAACATAT TATTCCGTAA AGGTAAAAAA 240TTCAATGAAA CACAAACACA CCAACAAGTT GGTAACATGT CCATAAACTA CGGAGCCAAC 300TTCCAACCAA ATGGTAATGC GTATTTATGC GTCTATGGTT GGACTGTTGA CCCTCTTGTC 360GAATATTATA TTGTCGACAG TTGGGGCAAC TGGCGTCCAC CAGGAGCAAC GCCTAAGGGG 420ACCATCACTG TTGATGGAGG AACATATGAT ATCTACGAGA CTCTTAGAGT CAATCAACCC 480TCCATTAAGG GGATTGCCAC ATTTAAACAA TATTGGAGTG TTCGAAGATC GAAACGCACG 540AGTGGCACGA TTTCTGTCAG CAACCACTTT AGAGCGTGGG AAAACTTAGG GATGAATATG 600GGGAAAATGT ATGAAGTCGC GCTTACTGTA GAAGGCTATC AAAGTAGCGG AAGTGCTAAT 660GTATATAGCA ATACACTAAG AATTAACGGT AACCCTCTCT CAACTATTAG TAATGACGAG 720AGCATAACTT TGGATAAAAA CAAT 744 744 base pairs nucleic acid single linearDNA (genomic) Bacillus CDS 1..744 mat_peptide 82..744 sig_peptide 1..815 ATG AGA CAA AAG AAA TTG ACG TTG ATT TTA GCC TTT TTA GTT TGT TTT 48 MetArg Gln Lys Lys Leu Thr Leu Ile Leu Ala Phe Leu Val Cys Phe -27 -25 -20-15 GCA CTA ACC TTA CCT GCA GAA ATA ATT CAG GCA CAA ATC GTC ACC GAC 96Ala Leu Thr Leu Pro Ala Glu Ile Ile Gln Ala Gln Ile Val Thr Asp -10 -5 15 AAT TCC ATT GGC AAC CAC GAT GGC TAT GAT TAT GAA TTT TGG AAA GAT 144Asn Ser Ile Gly Asn His Asp Gly Tyr Asp Tyr Glu Phe Trp Lys Asp 10 15 20AGC GGT GGC TCT GGG ACA ATG ATT CTC AAT CAT GGC GGT ACG TTC AGT 192 SerGly Gly Ser Gly Thr Met Ile Leu Asn His Gly Gly Thr Phe Ser 25 30 35 GCCCAA TGG AAC AAT GTT AAC AAC ATA TTA TTC CGT AAA GGT AAA AAA 240 Ala GlnTrp Asn Asn Val Asn Asn Ile Leu Phe Arg Lys Gly Lys Lys 40 45 50 TTC AATGAA ACA CAA ACA CAC CAA CAA GTT GGT AAC ATG TCC ATA AAC 288 Phe Asn GluThr Gln Thr His Gln Gln Val Gly Asn Met Ser Ile Asn 55 60 65 TAC GGA GCCAAC TTC CAA CCA AAT GGT AAT GCG TAT TTA TGC GTC TAT 336 Tyr Gly Ala AsnPhe Gln Pro Asn Gly Asn Ala Tyr Leu Cys Val Tyr 70 75 80 85 GGT TGG ACTGTT GAC CCT CTT GTC GAA TAT TAT ATT GTC GAC AGT TGG 384 Gly Trp Thr ValAsp Pro Leu Val Glu Tyr Tyr Ile Val Asp Ser Trp 90 95 100 GGC AAC TGGCGT CCA CCA GGA GCA ACG CCT AAG GGG ACC ATC ACT GTT 432 Gly Asn Trp ArgPro Pro Gly Ala Thr Pro Lys Gly Thr Ile Thr Val 105 110 115 GAT GGA GGAACA TAT GAT ATC TAC GAG ACT CTT AGA GTC AAT CAA CCC 480 Asp Gly Gly ThrTyr Asp Ile Tyr Glu Thr Leu Arg Val Asn Gln Pro 120 125 130 TCC ATT AAGGGG ATT GCC ACA TTT AAA CAA TAT TGG AGT GTT CGA AGA 528 Ser Ile Lys GlyIle Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Arg 135 140 145 TCG AAA CGCACG AGT GGC ACG ATT TCT GTC AGC AAC CAC TTT AGA GCG 576 Ser Lys Arg ThrSer Gly Thr Ile Ser Val Ser Asn His Phe Arg Ala 150 155 160 165 TGG GAAAAC TTA GGG ATG AAT ATG GGG AAA ATG TAT GAA GTC GCG CTT 624 Trp Glu AsnLeu Gly Met Asn Met Gly Lys Met Tyr Glu Val Ala Leu 170 175 180 ACT GTAGAA GGC TAT CAA AGT AGC GGA AGT GCT AAT GTA TAT AGC AAT 672 Thr Val GluGly Tyr Gln Ser Ser Gly Ser Ala Asn Val Tyr Ser Asn 185 190 195 ACA CTAAGA ATT AAC GGT AAC CCT CTC TCA ACT ATT AGT AAT GAC GAG 720 Thr Leu ArgIle Asn Gly Asn Pro Leu Ser Thr Ile Ser Asn Asp Glu 200 205 210 AGC ATAACT TTG GAT AAA AAC AAT 744 Ser Ile Thr Leu Asp Lys Asn Asn 215 220 248amino acids amino acid linear protein 6 Met Arg Gln Lys Lys Leu Thr LeuIle Leu Ala Phe Leu Val Cys Phe -27 -25 -20 -15 Ala Leu Thr Leu Pro AlaGlu Ile Ile Gln Ala Gln Ile Val Thr Asp -10 -5 1 5 Asn Ser Ile Gly AsnHis Asp Gly Tyr Asp Tyr Glu Phe Trp Lys Asp 10 15 20 Ser Gly Gly Ser GlyThr Met Ile Leu Asn His Gly Gly Thr Phe Ser 25 30 35 Ala Gln Trp Asn AsnVal Asn Asn Ile Leu Phe Arg Lys Gly Lys Lys 40 45 50 Phe Asn Glu Thr GlnThr His Gln Gln Val Gly Asn Met Ser Ile Asn 55 60 65 Tyr Gly Ala Asn PheGln Pro Asn Gly Asn Ala Tyr Leu Cys Val Tyr 70 75 80 85 Gly Trp Thr ValAsp Pro Leu Val Glu Tyr Tyr Ile Val Asp Ser Trp 90 95 100 Gly Asn TrpArg Pro Pro Gly Ala Thr Pro Lys Gly Thr Ile Thr Val 105 110 115 Asp GlyGly Thr Tyr Asp Ile Tyr Glu Thr Leu Arg Val Asn Gln Pro 120 125 130 SerIle Lys Gly Ile Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Arg 135 140 145Ser Lys Arg Thr Ser Gly Thr Ile Ser Val Ser Asn His Phe Arg Ala 150 155160 165 Trp Glu Asn Leu Gly Met Asn Met Gly Lys Met Tyr Glu Val Ala Leu170 175 180 Thr Val Glu Gly Tyr Gln Ser Ser Gly Ser Ala Asn Val Tyr SerAsn 185 190 195 Thr Leu Arg Ile Asn Gly Asn Pro Leu Ser Thr Ile Ser AsnAsp Glu 200 205 210 Ser Ile Thr Leu Asp Lys Asn Asn 215 220 81 basepairs nucleic acid single linear DNA (genomic) 7 ATGAGACAAA AGAAATTGACGTTGATTTTA GCCTTTTTAG TTTGTTTTGC ACTAACCTTA 60 CCTGCAGAAA TAATTCAGGC A81 81 base pairs nucleic acid single linear DNA (genomic) CDS 1..81sig_peptide 1..81 8 ATG AGA CAA AAG AAA TTG ACG TTG ATT TTA GCC TTT TTAGTT TGT TTT 48 Met Arg Gln Lys Lys Leu Thr Leu Ile Leu Ala Phe Leu ValCys Phe 1 5 10 15 GCA CTA ACC TTA CCT GCA GAA ATA ATT CAG GCA 81 Ala LeuThr Leu Pro Ala Glu Ile Ile Gln Ala 20 25 27 amino acids amino acidlinear protein 9 Met Arg Gln Lys Lys Leu Thr Leu Ile Leu Ala Phe Leu ValCys Phe 1 5 10 15 Ala Leu Thr Leu Pro Ala Glu Ile Ile Gln Ala 20 25 1513base pairs nucleic acid single linear DNA (genomic) Bacillus 10AAATTGAATT GTGTATATCT AATGATAACG ACAAATCGTC ACTGTTTTTA AACTAATCTC 60AAACCAATAC TTCTTTATTT AACGCTAACC ACTTGCAATC TTATCACAAG AACATTCTTT 120ATAGGAACTT TCCCATTTGC AAGACGATAA AAAATCTTTT TCCCCTATTT TATCTTATCG 180CCTTGATCGG TTTAATTTGT AAACTTTATT TTAGTTTACG TGATGTTCCC TCATTCATAC 240CATTAATCAC AGTTAACGCT AGAGTCATCT TTTTTCGGTT CTCAAAAATA CCTGAAGAAC 300ATTTATGTCA TATTTTCTCA CGCCGCTCCA TAATGGAATA TATATACTCT TTTATACATA 360TTAAGTAAAT TAGTATATAC TTGCGTTATC AAAATGTGAG ATAATCTAAT TGATCAAACA 420AGCAGCTATC CAAAAAACAC TGATGTTGAC CTCTTAAAGA AGTGTCACTA TCTATGAAAA 480GATAATTATC CAGTTTCAAA ATTTGAAATA GTGTGTATGG AATAGTTTGA ATGTCAACTG 540CTGTGAAAGG AGGGTAGGTA GTACCGTAGA CTTCATTACC AAAAATTAGT TGTAAAAAAA 600TTAAAAGGAG GAATGCCTAA TGAGACAAAA GAAATTGACG TTGATTTTAG CCTTTTTAGT 660TTGTTTTGCA CTAACCTTAC CTGCAGAAAT AATTCAGGCA CAAATCGTCA CCGACAATTC 720CATTGGCAAC CACGATGGCT ATGATTATGA ATTTTGGAAA GATAGCGGTG GCTCTGGGAC 780AATGATTCTC AATCATGGCG GTACGTTCAG TGCCCAATGG AACAATGTTA ACAACATATT 840ATTCCGTAAA GGTAAAAAAT TCAATGAAAC ACAAACACAC CAACAAGTTG GTAACATGTC 900CATAAACTAC GGAGCCAACT TCCAACCAAA TGGTAATGCG TATTTATGCG TCTATGGTTG 960GACTGTTGAC CCTCTTGTCG AATATTATAT TGTCGACAGT TGGGGCAACT GGCGTCCACC 1020AGGAGCAACG CCTAAGGGGA CCATCACTGT TGATGGAGGA ACATATGATA TCTACGAGAC 1080TCTTAGAGTC AATCAACCCT CCATTAAGGG GATTGCCACA TTTAAACAAT ATTGGAGTGT 1140TCGAAGATCG AAACGCACGA GTGGCACGAT TTCTGTCAGC AACCACTTTA GAGCGTGGGA 1200AAACTTAGGG ATGAATATGG GGAAAATGTA TGAAGTCGCG CTTACTGTAG AAGGCTATCA 1260AAGTAGCGGA AGTGCTAATG TATATAGCAA TACACTAAGA ATTAACGGTA ACCCTCTCTC 1320AACTATTAGT AATGACGAGA GCATAACTTT GGATAAAAAC AATTAAAAAT CCTTATCTCT 1380TTCGGTTCAG TTCTCATTAT TTTCAAATAA CCTCCCGGTT GGATCTTTTC CAACGGGAGG 1440TTTTATTGGA AAGGTTAAGT ATAGTATACT CCGATTCCAT CCAGAGGAAT GCTTGAAACA 1500CCTCCGTCAC TAG 1513 1513 base pairs nucleic acid single linear DNA(genomic) Bacillus CDS 620..1363 mat_peptide 701..1363 sig_peptide620..700 11 AAATTGAATT GTGTATATCT AATGATAACG ACAAATCGTC ACTGTTTTTAAACTAATCTC 60 AAACCAATAC TTCTTTATTT AACGCTAACC ACTTGCAATC TTATCACAAGAACATTCTTT 120 ATAGGAACTT TCCCATTTGC AAGACGATAA AAAATCTTTT TCCCCTATTTTATCTTATCG 180 CCTTGATCGG TTTAATTTGT AAACTTTATT TTAGTTTACG TGATGTTCCCTCATTCATAC 240 CATTAATCAC AGTTAACGCT AGAGTCATCT TTTTTCGGTT CTCAAAAATACCTGAAGAAC 300 ATTTATGTCA TATTTTCTCA CGCCGCTCCA TAATGGAATA TATATACTCTTTTATACATA 360 TTAAGTAAAT TAGTATATAC TTGCGTTATC AAAATGTGAG ATAATCTAATTGATCAAACA 420 AGCAGCTATC CAAAAAACAC TGATGTTGAC CTCTTAAAGA AGTGTCACTATCTATGAAAA 480 GATAATTATC CAGTTTCAAA ATTTGAAATA GTGTGTATGG AATAGTTTGAATGTCAACTG 540 CTGTGAAAGG AGGGTAGGTA GTACCGTAGA CTTCATTACC AAAAATTAGTTGTAAAAAAA 600 TTAAAAGGAG GAATGCCTA ATG AGA CAA AAG AAA TTG ACG TTG ATTTTA GCC 652 Met Arg Gln Lys Lys Leu Thr Leu Ile Leu Ala -27 -25 -20 TTTTTA GTT TGT TTT GCA CTA ACC TTA CCT GCA GAA ATA ATT CAG GCA 700 Phe LeuVal Cys Phe Ala Leu Thr Leu Pro Ala Glu Ile Ile Gln Ala -15 -10 -5 CAAATC GTC ACC GAC AAT TCC ATT GGC AAC CAC GAT GGC TAT GAT TAT 748 Gln IleVal Thr Asp Asn Ser Ile Gly Asn His Asp Gly Tyr Asp Tyr 1 5 10 15 GAATTT TGG AAA GAT AGC GGT GGC TCT GGG ACA ATG ATT CTC AAT CAT 796 Glu PheTrp Lys Asp Ser Gly Gly Ser Gly Thr Met Ile Leu Asn His 20 25 30 GGC GGTACG TTC AGT GCC CAA TGG AAC AAT GTT AAC AAC ATA TTA TTC 844 Gly Gly ThrPhe Ser Ala Gln Trp Asn Asn Val Asn Asn Ile Leu Phe 35 40 45 CGT AAA GGTAAA AAA TTC AAT GAA ACA CAA ACA CAC CAA CAA GTT GGT 892 Arg Lys Gly LysLys Phe Asn Glu Thr Gln Thr His Gln Gln Val Gly 50 55 60 AAC ATG TCC ATAAAC TAC GGA GCC AAC TTC CAA CCA AAT GGT AAT GCG 940 Asn Met Ser Ile AsnTyr Gly Ala Asn Phe Gln Pro Asn Gly Asn Ala 65 70 75 80 TAT TTA TGC GTCTAT GGT TGG ACT GTT GAC CCT CTT GTC GAA TAT TAT 988 Tyr Leu Cys Val TyrGly Trp Thr Val Asp Pro Leu Val Glu Tyr Tyr 85 90 95 ATT GTC GAC AGT TGGGGC AAC TGG CGT CCA CCA GGA GCA ACG CCT AAG 1036 Ile Val Asp Ser Trp GlyAsn Trp Arg Pro Pro Gly Ala Thr Pro Lys 100 105 110 GGG ACC ATC ACT GTTGAT GGA GGA ACA TAT GAT ATC TAC GAG ACT CTT 1084 Gly Thr Ile Thr Val AspGly Gly Thr Tyr Asp Ile Tyr Glu Thr Leu 115 120 125 AGA GTC AAT CAA CCCTCC ATT AAG GGG ATT GCC ACA TTT AAA CAA TAT 1132 Arg Val Asn Gln Pro SerIle Lys Gly Ile Ala Thr Phe Lys Gln Tyr 130 135 140 TGG AGT GTT CGA AGATCG AAA CGC ACG AGT GGC ACG ATT TCT GTC AGC 1180 Trp Ser Val Arg Arg SerLys Arg Thr Ser Gly Thr Ile Ser Val Ser 145 150 155 160 AAC CAC TTT AGAGCG TGG GAA AAC TTA GGG ATG AAT ATG GGG AAA ATG 1228 Asn His Phe Arg AlaTrp Glu Asn Leu Gly Met Asn Met Gly Lys Met 165 170 175 TAT GAA GTC GCGCTT ACT GTA GAA GGC TAT CAA AGT AGC GGA AGT GCT 1276 Tyr Glu Val Ala LeuThr Val Glu Gly Tyr Gln Ser Ser Gly Ser Ala 180 185 190 AAT GTA TAT AGCAAT ACA CTA AGA ATT AAC GGT AAC CCT CTC TCA ACT 1324 Asn Val Tyr Ser AsnThr Leu Arg Ile Asn Gly Asn Pro Leu Ser Thr 195 200 205 ATT AGT AAT GACGAG AGC ATA ACT TTG GAT AAA AAC AAT TAAAAATCCT 1373 Ile Ser Asn Asp GluSer Ile Thr Leu Asp Lys Asn Asn 210 215 220 TATCTCTTTC GGTTCAGTTCTCATTATTTT CAAATAACCT CCCGGTTGGA TCTTTTCCAA 1433 CGGGAGGTTT TATTGGAAAGGTTAAGTATA GTATACTCCG ATTCCATCCA GAGGAATGCT 1493 TGAAACACCT CCGTCACTAG1513 619 base pairs nucleic acid single linear DNA (genomic) 12AAATTGAATT GTGTATATCT AATGATAACG ACAAATCGTC ACTGTTTTTA AACTAATCTC 60AAACCAATAC TTCTTTATTT AACGCTAACC ACTTGCAATC TTATCACAAG AACATTCTTT 120ATAGGAACTT TCCCATTTGC AAGACGATAA AAAATCTTTT TCCCCTATTT TATCTTATCG 180CCTTGATCGG TTTAATTTGT AAACTTTATT TTAGTTTACG TGATGTTCCC TCATTCATAC 240CATTAATCAC AGTTAACGCT AGAGTCATCT TTTTTCGGTT CTCAAAAATA CCTGAAGAAC 300ATTTATGTCA TATTTTCTCA CGCCGCTCCA TAATGGAATA TATATACTCT TTTATACATA 360TTAAGTAAAT TAGTATATAC TTGCGTTATC AAAATGTGAG ATAATCTAAT TGATCAAACA 420AGCAGCTATC CAAAAAACAC TGATGTTGAC CTCTTAAAGA AGTGTCACTA TCTATGAAAA 480GATAATTATC CAGTTTCAAA ATTTGAAATA GTGTGTATGG AATAGTTTGA ATGTCAACTG 540CTGTGAAAGG AGGGTAGGTA GTACCGTAGA CTTCATTACC AAAAATTAGT TGTAAAAAAA 600TTAAAAGGAG GAATGCCTA 619 150 base pairs nucleic acid single linear DNA(genomic) 13 TAAAAATCCT TATCTCTTTC GGTTCAGTTC TCATTATTTT CAAATAACCTCCCGGTTGGA 60 TCTTTTCCAA CGGGAGGTTT TATTGGAAAG GTTAAGTATA GTATACTCCGATTCCATCCA 120 GAGGAATGCT TGAAACACCT CCGTCACTAG 150 56 base pairsnucleic acid single linear other nucleic acid /desc = “syntheticoligonucleotide” 14 CCCCCCTACG TAGCGGCCGC CCCGGCCGGT AACCTAGGAAGTCAGCGCCC TGCACC 56 56 base pairs nucleic acid single linear othernucleic acid /desc = “synthetic oligonucleotide” 15 CCCCCCTACGTAGGCCGGGG CGGCCGCGGT TACCTAGGGC CTCGTGATAC GCCTAT 56 31 base pairsnucleic acid single linear other nucleic acid /desc = “syntheticoligonucleotide” 16 ACGAGGAAAG ATGCTGTTCT TGTAAATGAG T 31 19 base pairsnucleic acid single linear other nucleic acid /desc = “syntheticoligonucleotide” 17 TACCTTGTCT ACAAACCCC 19 18 base pairs nucleic acidsingle linear other nucleic acid /desc = “synthetic oligonucleotide” 18CGGTCGCCGC ATACACTA 18 36 base pairs nucleic acid single linear othernucleic acid /desc = “synthetic oligonucleotide” 19 CCCCCCCCCGGTAACCTGCA TTAATGAATC GGCCAA 36 39 base pairs nucleic acid single linearother nucleic acid /desc = “synthetic oligonucleotide” 20 CCCCCCCCCGGTTACCGTAT TTATTAACTT CTCCTAGTA 39 50 base pairs nucleic acid singlelinear other nucleic acid /desc = “synthetic oligonucleotide” 21CCCCCCTCTA GATTAATTAA CCAAGCTTGG GATCCGTCGA CCTGCAGATC 50 39 base pairsnucleic acid single linear other nucleic acid /desc = “syntheticoligonucleotide” 22 CCCCCCTGAA ATCAGCTGGA CTAAAAGGGA TGCAATTTC 39 30base pairs nucleic acid single linear other nucleic acid /desc =“synthetic oligonucleotide” 23 CCCCCCGTCG ACCGCATGCG CCGGCACAGC 30 39base pairs nucleic acid single linear other nucleic acid /desc =“synthetic oligonucleotide” 24 CCCCCCGCAT GCGCAAATCG TCACCGACAATTCCATTGG 39 19 base pairs nucleic acid single linear other nucleic acid/desc = “synthetic oligonucleotide” 25 TACCTTGTCT ACAAACCCC 19 185 basepairs nucleic acid single linear DNA (genomic) 26 TCATGTAACT CGCCTTGATCTATTTCATTT GTATCAAAGG ATTTATACAC AAACAAGAGA 60 CATCCATGCC GGGTTAAAGCAGTATCGTTC CATCTAACAG AGAAGGNCTG CATGAAAGGA 120 GGTGATGGGT TTTTCATCTTAGGGATGACA GAACAATACG GATGAAAAAA GGAGAGGGAT 180 GGAAA 185 81 base pairsnucleic acid single linear DNA (genomic) 27 ATGAATTTGA AAAGATTGAGGCTGTTGTTT GTGATGTGTA TTGGATTTGT GCTGACACTG 60 ACGGCTGTGC CGGCTCATGC G81 81 base pairs nucleic acid single linear DNA (genomic) CDS 1..81 28ATG AAT TTG AAA AGA TTG AGG CTG TTG TTT GTG ATG TGT ATT GGA TTT 48 MetAsn Leu Lys Arg Leu Arg Leu Leu Phe Val Met Cys Ile Gly Phe 1 5 10 15GTG CTG ACA CTG ACG GCT GTG CCG GCT CAT GCG 81 Val Leu Thr Leu Thr AlaVal Pro Ala His Ala 20 25 27 amino acids amino acid linear protein 29Met Asn Leu Lys Arg Leu Arg Leu Leu Phe Val Met Cys Ile Gly Phe 1 5 1015 Val Leu Thr Leu Thr Ala Val Pro Ala His Ala 20 25

1. Xylanase, characterized in that it originates from a Bacillus strainand in that it is active over a pH range between approximately 5 and 10and over a temperature range between approximately 50 and 80° C. 2.Xylanase, characterized in that it originates from Bacillus sp. strain720/1 (LMG P-14798) or from a derivative or mutant of this strain. 3.Isolated and purified xylanase, characterized in that it comprises theamino acid sequence from 1 to 221 amino acids (SEQ ID NO:3) or amodified sequence derived from this sequence.
 4. Xylanase according toclaim 3, characterized in that it is synthesized in the form of aprecursor containing 248 amino acids (SEQ ID NO:6).
 5. Xylanase,characterized in that it consists of a single polypeptide having amolecular weight of approximately 25 kDa, and in that it has adetermined isoelectric point of between approximately 9.5 andapproximately 9.7.
 6. Xylanase, characterized in that it is producedheterologously by a microorganism of the genus Bacillus.
 7. An isolatedand purified culture of Bacillus sp. 720/1 (LMG P-14798) and culturederived or mutated from this culture.
 8. DNA molecule comprising thenucleotide sequence (SEQ ID NO:1) which codes for the mature xylanase ofBacillus sp. 720/1 (LMG P-14798) or a modified sequence derived fromthis sequence.
 9. DNA molecule according to claim 8, characterized inthat it comprises the nucleotide sequence (SEQ ID NO:4) which codes forthe Bacillus sp. 720/1 xylanase precursor or a modified sequence derivedfrom this sequence.
 10. DNA molecule according to claim 8 or 9,characterized in that it comprises the entire Bacillus sp. 720/1xylanase gene (SEQ ID NO:10).
 11. DNA molecule according to claim 8,characterized in that it comprises the promoter (SEQ ID NO:26) derivedfrom the gene which codes for Bacillus pumilus PRL B12 xylanase, thepresequence (SEQ ID NO:27) which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase and the nucleotide sequence (SEQ IDNO:1) which codes for Bacillus sp. 720/1 xylanase.
 12. Expression vectoror chromosomal integration vector containing the DNA molecule accordingto claim 8, 9, 10 or
 11. 13. Expression vector pUBRD-720X11. 14.Expression vector pBPXD-PRE-720X.
 15. Transformed strain comprising theDNA molecule according to claim 8, 9, 10 or
 11. 16. Transformed straincomprising the expression vector or the chromosomal integration vectoraccording to claim 12, 13 or
 14. 17. Transformed strain according toclaim 15 or 16, characterized in that it is a Bacillus strain. 18.Transformed strain according to claim 17, characterized in that it is aBacillus licheniformis or Bacillus pumilus strain.
 19. Xylanase producedby the transformed strain according to claim 15, 16, 17 or
 18. 20.Method for the production of a xylanase according to any one of claims 1to 6 or according to claim 19, characterized in that it comprises theculturing of a strain capable of producing xylanase in a suitablenutrient medium containing carbon and nitrogen sources and inorganicsalts under aerobic conditions, and the harvesting of the xylanasethereby obtained.
 21. Method for the preparation of a xylanase accordingto any one of claims 1 to 6, characterized in that it comprisesisolation of a DNA fragment coding for the xylanase, the insertion ofthis DNA fragment into a suitable vector, the introduction of thisvector into a suitable host or the introduction of this DNA fragmentinto the chromosome of a suitable host, the culturing of this host, theexpression of the xylanase and the harvesting of the xylanase.
 22. Useof a xylanase according to any one of claims 1 to 6 or according toclaim 19, for the treatment of paper pulp.
 23. Use of a xylanaseaccording to any one of claims 1 to 6 or according to claim 19 in animalfeeds.
 24. An enzyme composition containing a xylanase according to anyone of claims 1 to 6 or according to claim 19, and at least oneadditive.
 25. Promoter (SEQ ID NO:26) derived from the gene which codesfor Bacillus pumilus PRL B12 xylanase.
 26. Presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanase.27. Expression system which can be used for the production of apolypetide, characterized in that it comprises: the sequence of thepromoter (SEQ ID NO:26) derived from the gene which codes for Bacilluspumilus PRL B12 xylanase, a sequence coding for a signal peptide, andthe sequence of the polypeptide of interest.
 28. Expression system whichcan be used for the production of a polypeptide, characterized in thatit comprises: the sequence of a promoter, the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanase,and the sequence of the polypeptide of interest.
 29. Expression systemwhich can be used for the production of a polypeptide, characterized inthat it comprises: the sequence of the promoter (SEQ ID NO:26) derivedfrom the gene which codes for Bacillus pumilus PRL B12 xylanase, thepresequence (SEQ ID NO:27) which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase, the sequence of the polypeptide ofinterest, and the sequence of a terminator.
 30. Expression systemaccording to claim 27, 28 or 29, characterized in that the sequence ofthe polypeptide of interest corresponds to the nucleotide sequence (SEQID NO:1) which codes for Bacillus sp. 720/1 xylanase.